JP4426094B2 - α-amylase mutant - Google Patents

Abstract

The invention relates to a variant of a parent Termamyl-like ±-amylase, which exhibits an alteration in at least one of the following properties relative to said parent ±-amylase: i) improved pH stability at a pH from 8 to 10.5; and/or ii) improved Ca 2+ stability at pH 8 to 10.5, and/or iii) increased specific activity at temperatures from 10 to 60°C.

Description

【００８２】
式中、ｘ_i は、当プログラムによって計算して得られた、アミノ酸及びアミノ酸の基の量であり、ｙ_i は、当プログラムの使用者によって決められた、アミノ酸及びアミノ酸の基の必要とされる量であり（例えば、通常の20アミノ酸又は停止コドンのうちのいずれが、例えばある割合（例えば90％ Ala, ３％ Ile, ７％ Val) にて、導入されることが必要とされるかを特定する）、そしてｗ_i は、当プログラムの使用者によって決められた、割当の荷重係数である（例えば、問題の位置に挿入される特定のアミノ酸残基を有することの重要性に依存する）。Ｎは、当プログラムの使用者によって決められたアミノ酸基の数＋21である。この関数のために、０⁰ を１とする。
【００８３】
この関数の最大値を見い出すために、Monte-Carlo アルゴリズム（１つの例が、Valleau, J.P. & Whittington, S.G. (1977) A guide to Mont Carlo for statistical mechanics : 1 Highways 。In“Stastistical Mechanics, Part A”Equlibrium Techniqeues ed. B.J. Berne, New York : Plenumに記載されている）を用いる。各反復において、以下の過程が行われる：
１．各塩基において、新しいランダムなヌクレオチド組成が選択され、その場合、その時の組成と新しい組成との間の絶対差が、当コドンの３つの全ての位置が、４つのヌクレオチドＧ，Ａ，Ｔ，Ｃの各々である場合のｄよりも小さいか、又は等しい（ｄの定義は下記参照のこと）。
２．新しい組成の評価点と、その時の組成の評価点とを、前記の関数ｓを用いて比較する。新しい評価点が、その時の評価点よりも大きいか、又は等しい場合には、新しい組成を保持し、その時の組成を新しい組成に変える。新しい評価点がより小さい場合には、新しい組成を保持する確率は、exp [1000 [新しい評価点−その時の評価点]]である。
【００８４】
１サイクルは、標準的には、前記の反復過程1000回から成り、その場合、ｄは、１から０へ直線的に減少する。最適化においては、 100回以上のサイクルを行う。最高の評価点を得たヌクレオチド組成が、最後に提示される。
〔実施例〕
実施例１：
Termamyl の相同性構築の例
B. licheniformisαアミラーゼ（以下Termamylとする）の、その他のTermamyl様αアミラーゼに対する総体的な相同性は高く、そしてその相似率は極端に高い。GCG プログラム（University of Wisconsin Genetics Computer Group's program)を用いて計算された、 BSG（配列番号３を有するB. stearothermophilus αアミラーゼ）、及び BAN（配列番号５を有するB. awyloliquefaciensαアミラーゼ）に対するTermamylの相似率は、各々89％及び78％であった。Termamylは、BAN 及びBSG と比べて、残基G180とK181との間の２残基が欠失している。BSG は、BAN 及びTermamylと比べて、G371とI372との間の３残基が欠失している。Ｎ末端において、BSG と比べて、BAN は２残基少く、そしてTermamylは１残基少い。
【００８５】
B. licheniformisαアミラーゼ（Termamyl）及びB. awyloliquefaciensαアミラーゼ(BAN) の各構造のモデルが、WO96/23974の付表１に開示されている構造に基づいて構築された。他のTermamyl様αアミラーゼ（例えば本明細書に開示したもの）の構造も、同様なやり方で構築し得る。
構造を解明するために用いられるαアミラーゼと比べて、Termamylは、 178〜182 付近の２つの残基が欠失している点で、異なっている。モデル構造において、これを補うために、BIOSYMのHOMOLOGYプログラムを用いて、欠失点を除いて、当構造の相当する位置（構造的に保存された領域だけでなく）における残基を置換した。Termamyl（BAN)におけるG179 (G177) とK180 (K180) との間でペプチド結合を成立させた。解明された構造とモデル構造との間の構造上の密接な関係（従って後者の有効性）が、モデルにおいて相互にあまりに近接していることが判明した原子が、非常にほんの少ししか存在しないことから指摘される。
【００８６】
次にINSIGHT プログラムによって、この非常に粗いTermamylの構造に、解明された構造の場合（WO96/23874の付表１参照）と同じ座標で、その解明された構造に由来する全ての水(605個）及びイオン（４カルシウムと１ナトリウム）を付け加えた。この場合、ほんの少しの重複しか認められず、すなわち非常に良好な適合が認められた。次に、このモデル構造を、200 過程の急勾配降下（Steepest descent) 及び600 過程の共役勾配（Conjugated gradient)によって最小化した（Brooks et al., 1983, J. Computational Chemistry 4, p. 187-217)。次に、この最小化構造に分子動力学を適用し、35psを超えて 200psの平衡まで、５psの加熱を行った。この動力学は、Verletアルゴリズムに従って運用され、Behrendsenによる水環境との共役（Berendsen et al., 1984, J. Chemical Physics 81, p. 3684-3690) に従って平衡温度 300Ｋを維持した。ピコ秒毎に回転と並進を取り出した。
実施例２：
pH 安定性の向上及び温度依存的な活性の変化を示すαアミラーゼ変異体を同定するために重要な領域を導き出す方法
注目の酵素、ここではSP722 とTermamyl、のＸ線解析構造及び／又はモデル構築構造に対して、分子動力学的シュミレーションを行う。この分子動力学的シュミレーションを、CHARMM (Molecular simulations (MSI））プログラム又はその他の適当なプログラム、例えば DISCOVER (MSI) によって行う。この分子動力学的分析を、孤立状態、あるいは、より好ましくは、結晶水を含んだ状態、又は、水中に、例えば水球若しくは水箱中に埋め込まれた状態、の酵素に対して行う。このシュミレーションを、 300ピコ秒（ps）又はそれ以上、例えば 300〜1200psの期間運用する。前記構造のＣα炭素に関して、等方性振動を取り出し、前記構造間で比較する。一方の配列中に欠失及び／又は挿入がある場合、他方の構造に由来する等方性振動をはめ込み、等方性変動の差を０にする。等方性振動の説明に関して、CHARMMの使用説明書（MSI から入手できる）を参照すること。
【００８７】
荷電性アミノ酸に対する標準的な荷電に従って、この分子動力学的シュミレーションを行うことができる。すなわち、Asp 及びGlu は陰性荷電され、そしてLys 及びArg は陽性荷電される。この条件は、約７の中性pHを近似する。より高い又は低いpHで分析するために、標準滴定可能な残基の変化したpKa を通常pH２〜10の範囲内で得て、当分子の滴定を行うことができる。この様な残基は、Lys, Arg, Asp, Glu, Tyr 及びHis である。また、Set, Thr及びCys は滴定可能であるが、ここでは考慮しない。ここで、pHのために変化した荷電に関して、高pHでは、Asp 及びGlu は陰性であり、Arg 及びLys は非荷電である。これは、約10〜11のpHを近似していて、この場合、Lys 及びArg の標準pKa が約９〜11であるとして、これらの残基の滴定を開始する。
１．高pH安定性を有するαアミラーゼ変異体を同定するために重要な領域を導き出すために用いる方法：
pH安定性が改良された変異体を構築するために重要な領域は、極端なpHにおいて最良の運動性を示す領域、すなわち最大の等方性振動を有する領域である。
【００８８】
この様な領域は、以下の２つの分子動力学シュミレーションを行うことによって同定される：ｉ）塩基性アミノ酸Lys 及びArg が中性であり（すなわちプロトン化されていない）、そして酸性アミノ酸Asp 及びGlu が荷電（−１）を有する高pHでの運用、並びに、ii）塩基性アミノ酸Lys 及びArg が正味の荷電（＋１）を有し、そして前記酸性アミノ酸が荷電（−１）を有する中性pHでの運用。
【００８９】
この２つの運用を比較して、中性pHに比べて高pHにて相対的により高い運動性を示す領域を同定した。
一般的な安定性、例えば水素結合、を向上させる残基、その領域をより硬直にする（例えば、グリシン残基のプロリン置換などの変異による）残基、あるいは、荷電又は残基間相互作用を向上させる残基を導入することによって、当酵素の高pH安定性が向上する。
２．中温での活性が増加したαアミラーゼ変異体を同定するための領域を取り出す方法：
中温での活性が増加した変異体を構築するために重要な領域を、SP722 とTermamylとの等方性振動の差、すなわちSP722 でのＣαの等方性振動からTermamylでのものを引いた差から、見い出した。その等方性振動の運動性が最大である領域を選択した。この様な領域及びそこの残基が、中温での活性を増加させると期待された。それらの残基に適切な運動性が存在する場合には、αアミラーゼの活性が発現するだけである。それらの残基の運動性があまりに低い場合、活性は低下又は消滅する。
実施例３：
局所的ランダム dope 変異誘発法による、親酵素と比べて中温での Ca ²⁺ 安定性が向上した Termamyl 様αアミラーゼの構築
低カルシウム濃度におけるαアミラーゼの安定性を改良するために、予じめ選択した領域で、ランダム変異誘発を行った。
領域：SAI
残基：R181-W189
停止コドンの量を最小化する様に、SAI 領域において示唆された各変異のための導入（spiked）コドンを決定するために、DOPEプログラム（「材料と方法」参照）を用いた（表１）。示唆された一群のアミノ酸変異を得るために、そのコドンの３つの位置において、ヌクレオチドの正確な分布を計算した。希望の残基にする確率を高くするために、指示された位置において特異的に対象領域を処理したが、それでもなお他の可能性もあり得る。
表１：
各位置におけるアミノ酸残基の分布

表２に、得られた処理済みオリゴヌクレオチド鎖をセンス鎖として示し、それと共に、野生型のヌクレオチド配列及びアミノ酸配列、並びに各処理位置におけるヌクレオチド分布も示す。
表２：

ランダム変異誘発
表２から明白な導入（spiked）オリゴヌクレオチド（FSA とする）及びSAI 領域のための逆方向プライマーRSA 、並びにSacII 部位からDraII 部位までのSP722 （配列番号２）特異的プライマーを用いて、21塩基対の重複による重複伸長方法（Horton et al., Gene 77 (1989), pp. 61-68) によって、PCR ライブラリー断片を作成する。このPCR の鋳型は、プラスミドpJE1である。このPCR 断片を、大腸菌／バチルス菌シャトルベクターpDorK101にクローン化する（「材料と方法」参照）ことで、大腸菌で変異誘発し、即座にバチルス・ズブチリスで発現させることが可能となり、従って大腸菌内でのアミラーゼの致死的な集積を回避する。大腸菌内でクローン化PCR 断片を確立した後、そのプラスミドから修飾されたpUC19 断片を切り出し、そしてプロモーターと、変異Termamyl遺伝子とを自然に連結し、その様にしてバチルス菌内での発現が可能となる。
スクリーニング
「材料と方法」で記載した通り、このライブラリーを低カルシウムフィルター検査でスクリーニングし得る。
実施例４：
配列番号１のアミラーゼ (SP690) の変異体の構築
配列番号１のアミラーゼをコードする遺伝子は、WO96/23873に記載されているプラスミドpTVB106 中に存在する。バチルス・ズブチリス内で、この構成体のamyLプロモーターから、そのアミラーゼを発現させる。
【００９０】
当タンパク質の１つの変異体は、デルタ（T183−G184）＋Y243F ＋Q391E ＋K444Q である。この変異体の構築は、WO96/23873に記載されている。
Sarkar and Sommer, (1990), BioTechniques 8 : 404-407に記載されたメガプライマー法によるデルタ（T183−G184) ＋N195F の構築を以下に示す。
遺伝子特異的プライマーＢ１（配列番号17）及び変異生成プライマー101458（配列番号19）を用いて、pTVB106 様プラスミド（配列番号１のアミラーゼをコードする遺伝子中にデルタ（T183−G184）変異を有するもの）から、約 645bpのDNA 断片を、PCR によって増幅した。
【００９１】
この 645bp断片をアガロースゲルから精製し、これをメガプライマーとして、プライマーＹ２（配列番号18）と共に用いて、同一の鋳型に対して２回目のPCR を行った。
生じた約1080bpの断片を、制限酵素BstEIIとAflIIIとで切断し、生じた約 510bpのDNA 断片を精製し、同酵素で切断したpTVB106 様プラスミド（配列番号１のアミラーゼをコードする遺伝子中にデルタ（T183−G184）変異を有するもの）に連結した。この連結物によって、コンピテントなバチルス・ズブチリスSHA273細胞を形質転換し、そのクロラムフェニコール耐性の形質転換体を、DNA 配列決定によって検査して、プラスミド上に適切な変異が存在することを確認した。
プライマーＢ１：（配列番号17）
5' CGA TTG CTG ACG CTG TTA TTT GCG 3'
プライマーＹ２：（配列番号18）
5' CTT GTT CCC TTG TCA GAA CCA ATG 3'
プライマー101458：（配列番号19）：
5' GT CAT AGT TGC CGA AAT CTG TAT CGA CTT C 3'
変異体デルタ（T183−G184）＋K185R ＋A186T を、変異生成プライマー101638を用いること以外は、先と同様にして構築した。
プライマー101638：（配列番号20）
5' CC CAG TCC CAC GTA CGT CCC CTG AAT TTA TAT ATT TTG 3'
変異体：デルタ（T183−G184）＋A186T 、デルタ（T183−G184）＋A186I 、デルタ（T183−G184）＋A186S 、デルタ（T183−G184）＋A186N を、pTVB106 様プラスミド（変異体デルタ（T183−G184）＋K185R ＋A186T を有する）を鋳型及びクローニングベクターとして用いること以外は、先と同様にして構築する。そのための変異生成オリゴヌクレオチド（オリゴ１）は、
5' CC CAG TCC CAG NTCTTT CCC CTG AAT TTA TAT ATT TTG 3' （配列番号21）
である。
【００９２】
Ｎは、この変異生成オリゴヌクレオチドの合成時に、４つの塩基：Ａ，Ｃ，Ｇ及びＴの混合物を使用したことを意味する。形質転換体のDNA 配列決定によって、その成熟タンパク質のアミノ酸位置186 における適正なコドンを確認する。
変異体デルタ（T183−G184）＋K185R ＋A186T ＋N195F を以下の様にして構築する。
【００９３】
pTVB106 様プラスミド（デルタ（T183−G184）＋K185R ＋A186T の変異を有するもの）に対してプライマーｘ２（配列番号22）及びプライマー101458（配列番号19）を用いてPCR を行う。できたDNA 断片をメガプライマーとして、プライマーＹ２（配列番号18）と共に用いて、pTVB106 様プラスミド（デルタ（T183−G184）＋N195F の変異を有するもの）に対してPCR を行う。この２回目のPCR 産物を、制限エンドヌクレアーゼAcc65I及びAflIIIによって切断し、同酵素によって切断したpTVB106 様プラスミド（デルタ（T183−G184）＋N195F)中にクローン化した。
プライマーｘ２：（配列番号22）
5' GCG TGG ACA AAG TTT GAT TTT CCT G 3'
変異体：デルタ（T183−G184）＋K185R ＋A186T ＋N195F ＋Y243F ＋Q391E ＋K444Q を以下の様にして構築する。
【００９４】
pTVB106 様プラスミド（デルタ（T183−G184）＋K185R ＋A186T の変異を有するもの）に対してプライマーｘ２及びプライマー101458を用いて、PCR を行う。できたDNA 断片をメガプライマーとして、プライマーＹ２と共に用いて、pTVB106 様プラスミド（デルタ（T183−G184）＋Y243F ＋Q391E ＋K444Q)の変異を有するもの）に対してPCR を行う。この２回目のPCR 産物を、制限エンドヌクレアーゼAcc65I及びAflIIIによって切断し、同酵素によって切断したpTVB106 様プラスミド（デルタ（T183−G184）＋Y243F ＋Q391E ＋K444Q)中にクローン化した。
実施例５
親酵素 SP722 αアミラーゼ（配列番号２）における部位特異的αアミラーゼ変異体の構築
配列番号２のアミラーゼ(SP722）の変異体を以下の様にして構築する。
【００９５】
配列番号２のアミラーゼをコードする遺伝子は、WO96/23873に記載のプラスミドpTVB112 中に存在する。このアミラーゼは、バチルス・ズブチリス内で、この構成体のamyLプロモーターから発現される。
Sarkar and Sommer, 1990 (BioTechniques 8 : 404-407) に記載されたメガプライマー法によるデルタ（D183−G184）＋V56Iの構築を以下に示す。
【００９６】
遺伝子特異的プライマーDA03及びメガプライマーDA07を用いて、pTVB112 様プラスミド（配列番号２のαアミラーゼをコードする遺伝子中にデルタ（D183−G184）の変異を有するもの）から、約 820bpのDNA 断片をPCR によって増幅する。
この 820bp断片をアガロースゲルから精製し、これをメガプライマーとして、プライマーDA01と共に用いて、同一の鋳型に対して２回目のPCR を行う。
【００９７】
生じた約 920bpの断片を、制限酵素NgoMI 及びAatII によって切断し、生じた約 170bpのDNA 断片を精製し、同酵素で切断したpTVB112 様プラスミド（配列番号２のアミラーゼをコードする遺伝子中にデルタ（D183−G184）変異を有するもの）に連結する。この連結物によって、コンピテントなバチルス・ズブチリスSHA273細胞（低アミラーゼ及び低プロテアーゼ）を形質転換し、そのクロラムフェニコール耐性の形質転換体を、DNA 配列決定によって検査して、プラスミド上に適切な変異が存在することを確認する。
プライマーDA01：（配列番号23）
5' CCTAATGATGGGAATCACTGG 3'
プライマーDA03：（配列番号24）
5' GCATTGGATGCTTTTGAACAACCG 3'
プライマーDA07：（配列番号25）
5' CGCAAAATGATATCGGGTATGGAGCC 3'
変異体：デルタ（D183−G184）＋K108L 、デルタ（D183−G184）＋K108Q 、デルタ（D183−G184）＋K108E 、デルタ（D183−G184）＋K108V を、Sarkar and Sommer, 1990 (BioTechniques 8 : 404-407) に記載されたメガプライマー法によって構築した。
【００９８】
プライマーDA03及び変異生成プライマーDA20を用いてpTVB112 様プラスミド（デルタ（D183−G184）の変異を有するもの）に対してPCR を行う。生じたDNA 断片をメガプライマーとして、プライマーDA01と共に用いて、pTVB112 様プラスミド（デルタ（D183−G184）の変異を有するもの）に対してPCR を行う。２回目のPCR で生じた約 920bpの断片を、制限酵素AatII 及びMluIによって切断し、同酵素で切断したpTVB112 様プラスミド（デルタ（D183−G184）変異を有するもの）に連結する。
プライマーDA20：（配列番号26）：
5' GTGATGAACCACSWAGGTGGAGCTGATGC 3'
Ｓは、変異生成オリゴヌクレオチドを合成する際に、２つの塩基Ｃ及びＧの混合物を用いること、そしてＷは、その際に、２つの塩基Ａ及びＴの混合物を用いることを意味する。形質転換体の配列決定によって、成熟アミラーゼのアミノ酸位置108 に適切なコドンが存在することを確認する。
【００９９】
変異体：デルタ（D183−G184）＋D168A 、デルタ（D183−G184）＋D168I 、デルタ（D183−G184）＋D168V 、デルタ（D183−G184）＋D168T を、変異生成プライマーDA14を用いること以外は同様にして、構築する。
プライマーDA14（配列番号27）：
5' GATGGTGTATGGRYCAATCACGACAATTCC 3'
Ｒは、変異生成オリゴヌクレオチドを合成する際に、２つの塩基Ａ及びＧの混合物を用いること、そしてＹは、その際に、２つの塩基Ｃ及びＴの混合物を用いることを意味する。形質転換体の配列決定によって、成熟アミラーゼのアミノ酸位置168 に適切なコドンが存在することを確認する。
【０１００】
変異体：デルタ（D183−G184）＋Q169N を、変異生成プライマーDA15を用いること以外は同様にして構築する。
プライマーDA15（配列番号28）：
5' GGTGTATGGGATAACTCACGACAATTCC 3'
変異体：デルタ（D183−G184）＋Q169L を、変異生成プライマーDA16を用いること以外は同様にして構築する。
プライマーDA16（配列番号29）：
5' GGTGTATGGGATCTCTCACGACAATTCC 3'
変異体：デルタ（D183−G184）＋Q172N を、変異生成プライマーDA17を用いること以外は同様にして構築する。
プライマーDA17（配列番号30）：
5' GGGATCAATCACGAAATTTCCAAAATCGTATC 3'
変異体：デルタ（D183−G184）＋Q172L を、変異生成プライマーDA18を用いること以外は同様にして構築する。
プライマーDA18（配列番号31）：
5' GGGATCAATCACGACTCTTCCAAAATCGTATC 3'
変異体：デルタ（D183−G184）＋L201I を、変異生成プライマーDA06を用いること以外は同様にして構築する。
プライマーDA06（配列番号32）：
5' GGAAATTATGATTATATCATGTATGCAGATGTAG 3'
変異体：デルタ（D183−G184）＋K269S を、変異生成プライマーDA09を用いること以外は同様にして構築する。
プライマーDA09（配列番号33）：
5' GCTGAATTTTGGTCGAATGATTTAGGTGCC 3'
変異体：デルタ（D183−G184）＋K269Q を、変異生成プライマーDA11を用いること以外は同様にして構築する。
プライマーDA11（配列番号34）：
5' GCTGAATTTTGGTCGAATGATTTAGGTGCC 3'
変異体：デルタ（D183−G184）＋N270Y を、変異生成プライマーDA21を用いること以外は同様にして構築する。
プライマーDA21（配列番号35）：
5' GAATTTTGGAAGTACGATTTAGGTCGG 3'
変異体：デルタ（D183−G184）＋L272A 、デルタ（D183−G184）＋L272I 、デルタ（D183−G184）＋L272V 、デルタ（D183−G184）＋L272T を、変異生成プライマーDA12を用いること以外は同様にして構築する。
プライマーDA12（配列番号36）：
5' GGAAAAACGATRYCGGTGCCTTGGAGAAC 3'
Ｒは、変異生成オリゴヌクレオチドを合成する際に、２つの塩基Ａ及びＧの混合物を用いること、そしてＹは、その際に、２つの塩基Ｃ及びＴの混合物を用いることを意味する。形質転換体の配列決定によって、成熟アミラーゼのアミノ酸位置272 に適切なコドンが存在することを確認する。
【０１０１】
変異体：デルタ（D183−G184）＋L275A 、デルタ（D183−G184）＋L275I 、デルタ（D183−G184）＋L275V 、デルタ（D183−G184）＋L275T を、変異生成プライマーDA13を用いること以外は同様にして構築する。
プライマーDA13（配列番号37）：
5' GATTTAGGTGCCTRYCAGAACTATTTA 3'
Ｒは、変異生成オリゴヌクレオチドを合成する際に、２つの塩基Ａ及びＧの混合物を用いること、そしてＹは、その際に、２つの塩基Ｃ及びＴの混合物を用いることを意味する。形質転換体の配列決定によって、成熟アミラーゼのアミノ酸位置275 に適切なコドンが存在することを確認する。
【０１０２】
変異体：デルタ（D183−G184）＋Y295E を、変異生成プライマーDA08を用いること以外は同様にして構築する。
プライマーDA08（配列番号38）：
5' CCCCCTTCATGAGAATCTTTATAACG 3'
Sarkar and Sommer, 1990 (BioTechniques 8 : 404-407) に記載されたメガプライマー法によるデルタ（D183−G184）＋K446Q の構築を以下に示す。
【０１０３】
停止コドンの下流 214〜231bp にアニールする遺伝子特異的プライマーDA04及び変異生成プライマーDA10を用いて、pTVB112 様プラスミド（配列番号２のアミラーゼをコードする遺伝子中にデルタ（D183−G184）の変異を有するもの）から、約 350bpのDNA 断片をPCR によって増幅した。
生じたDNA 断片をメガプライマーとして、プライマーDA05と共に用いて、pTVB112 様プラスミド（デルタ（D183−G184）の変異を有するもの）に対してPCR を行う。この２回目のPCR による約 460bpの産物を、制限酵素SnaBI 及びNotIによって切断し、そして同酵素によって切断されたpTVB112 様プラスミド（デルタ（D183−G184））中にクローン化する。
プライマーDA04（配列番号39）：
5' GAATCCGAACCTCATTACACATTCG 3'
プライマーDA05（配列番号40）：
5' CGGATGGACTCGAGAAGGAAATACCACG 3'
プライマーDA10（配列番号41）：
5' CGTAGGGCAAAATCAGGCCGGTCAAGTTTGG 3'
変異体：デルタ（D183−G184）＋K458R を、変異生成プライマーDA22を用いること以外は同様にして構築する。
プライマーDA22（配列番号42）：
5' CATAACTGGAAATCGCCCGGGAACAGTTACG 3'
変異体：デルタ（D183−G184）＋P459S 及びデルタ（D183−G184）＋P459T を、変異生成プライマーDA19を用いること以外は同様にして構築する。
プライマーDA19（配列番号43）：
5' CTGGAAATAAAWCCGGAACAGTTACG 3'
Ｗは、変異生成プライマーを合成する際に、２つの塩基Ａ及びＴの混合物を用いることを意味する。形質転換体の配列決定によって、成熟アミラーゼのアミノ酸位置459 に適切なコドンが存在することを確認する。
【０１０４】
変異体：デルタ（D183−G184）＋T461P を、変異生成プライマーDA23を用いること以外は同様にして構築する。
プライマーDA23（配列番号44）：
5' GGAAATAAACCAGGACCCGTTACGATCAATGC 3'
変異体：デルタ（D183−G184）＋K142R を、変異生成プライマーDA32を用いること以外は同様にして構築する。
プライマーDA32（配列番号45）：
5' GAGGCTTGGACTAGGTTTGATTTTCCAG 3'
変異体：デルタ（D183−G184）＋K269R を、変異生成プライマーDA31を用いること以外は同様にして構築する。
プライマーDA31（配列番号46）：
5' GCTGAATTTTGGCGCAATGATTTAGGTGCC 3'
実施例６
親の Termamyl αアミラーゼ（配列番号４）における部位特異的αアミラーゼ変異体の構築
TermamylαアミラーゼをコードするamyL遺伝子は、WO95/10603 (Novo Nordisk) に記載されたプラスミドpDN1528 中に存在する。この親に各々が由来する置換変異体N265R 及びN265D を、WO97/41213に記載の方法又は前記の「メガプライマー」法によって構築する。変異生成プライマーは、以下の通りである：
N265R 置換のためのプライマーｂ11：
5' PCC AGC GCG CCT AGG TCA CGC TGC CAA TAT TCA G (配列番号56）
N265D 置換のためのプライマーｂ12：
5' PCC AGC GCG CCT AGG TCA TCC TGC CAA TAT TCA G (配列番号57）
Ｐはリン酸基を表す。
実施例７
配列番号２のアミノ酸配列を有する親αアミラーゼに由来する変異体におけるアルカリ pH での pH 安定性の決定
この一連の分析では、精製酵素試料を用いた。pH10.5に合わせた100mM CAPS緩衝液による各変異体溶液を用いて、測定を行った。この溶液を75℃でインキュベーションした。
【０１０５】
20分及び30分間インキュベーションした後、PNP-G7検査（「材料と方法」参照）によって残存活性を測定した。試料中の残存活性を、Britton Robinson緩衝液(pH7.3) を用いて測定した。高pH及び75℃でインキュベーションしなかった同酵素の対応参照溶液の０分時点と比較して、残存活性の減少を測定した。
以下の表では、親酵素（配列番号２）及び問題の変異体において、初期活性の関数として、それに対する比率％を示している。
【０１０６】

もう１つの一連の分析では、培養上清を用いた。pH10.5に合わせた100mM CAPS緩衝液による各変異体溶液を用いて、測定を行った。この溶液を80℃でインキュベーションした。
【０１０７】
30分間インキュベーションした後、Phadebas検査（「材料と方法」参照）によって、残存活性を測定した。試料中の残存活性を、Britton Robinson緩衝液(pH7.3) を用いて測定した。高pH及び80℃でインキュベーションしなかった同酵素の対応参照溶液の０分時点と比較して、残存活性の減少を測定した。
以下の表では、親酵素（配列番号２）及び問題の変異体において、初期活性の関数として、それに対する比率％を示している。
【０１０８】

実施例８
配列番号１，２及び４のアミノ酸配列を有する親αアミラーゼに由来する変異体におけるアルカリ pH でのカルシウム安定性の決定
Ａ．配列番号１の配列の変異体におけるカルシウム安定性
pH10.5に合わせた100mM CAPS緩衝液による各変異体溶液を用いて、測定を行った。この溶液に（ｔ＝０時点で）終濃度 2400ppmになる様にポリリン酸を加えた。この溶液を50℃でインキュベーションした。
【０１０９】
20分及び30分間インキュベーションした後、PNP-G7検査（前記）によって、残存活性を測定した。試料中の残存活性を、Britton Robinson緩衝液(pH7.3) を用いて測定した。高pH及び50℃でインキュベーションしなかった同酵素の対応参照溶液の０分時点と比較して、残存活性の減少を測定した。
以下の表では、親酵素（配列番号１）及び問題の変異体において、初期活性の関数として、それに対する比率％を示している。
【０１１０】

n.d.＝未決定
Ｂ．配列番号２の配列の変異体におけるカルシウム安定性
この一連の分析では、精製酵素試料を用いた。pH10.5に合わせた100mM CAPS緩衝液による各変異体溶液を用いて、測定を行った。この溶液に（ｔ＝０時点で）終濃度 2400ppmになる様にポリリン酸を加えた。この溶液を50℃でインキュベーションした。
【０１１１】
20分及び30分間インキュベーションした後、PNP-G7検査（前記）によって、残存活性を測定した。試料中の残存活性を、Britton Robinson緩衝液(pH7.3) を用いて測定した。高pH及び50℃でインキュベーションしなかった同酵素の対応参照溶液の０分時点と比較して、残存活性の減少を測定した。
以下の表では、親酵素（配列番号２）及び問題の変異体において、初期活性の関数として、それに対する比率％を示している。
【０１１２】

もう１つの一連の分析では、培養上清を用いた。pH10.5に合わせた100mM CAPS緩衝液による各変異体溶液を用いて、測定を行った。この溶液に（ｔ＝０時点で）終濃度 2400ppmになる様にポリリン酸を加えた。この溶液を50℃でインキュベーションした。
【０１１３】
30分間インキュベーションした後、前記のPhadebas検査によって、残存活性を測定した。試料中の残存活性を、Britton Robinson緩衝液(pH7.3) を用いて測定した。高pH及び50℃でインキュベーションしなかった同酵素の対応参照溶液の０分時点と比較して、残存活性の減少を測定した。
以下の表では、親酵素（配列番号２）及び問題の変異体において、初期活性の関数として、それに対する比率％を示している。
【０１１４】

Ｃ．配列番号４の配列の変異体におけるカルシウム安定性
pH10.5に合わせた100mM CAPS緩衝液による各変異体溶液を用いて、測定を行った。この溶液に（ｔ＝０時点で）終濃度 2400ppmになる様にポリリン酸を加えた。この溶液を60℃でインキュベーションした。
【０１１５】
20分間インキュベーションした後、PNP-G7検査（前記）によって、残存活性を測定した。試料中の残存活性を、Britton Robinson緩衝液(pH7.3) を用いて測定した。高pH及び60℃でインキュベーションしなかった同酵素の対応参照溶液の０分時点と比較して、残存活性の減少を測定した。
以下の表では、親酵素（配列番号４）及び問題の変異体において、初期活性の関数として、それに対する比率％を示している。
【０１１６】

実施例９
配列番号１のアミノ酸配列を有するαアミラーゼにおける中温での活性の測定
Ａ．配列番号１の配列の変異体におけるαアミラーゼ活性
pH7.3 に合わせた50mM Britton Robinson 緩衝液による各変異体溶液を用いて、前記のPhadebas検査によって測定を行った。試料中の活性を、50mM Britton Robinson 緩衝液(pH7.3) を用いて37℃で、そして50mM CAPS 緩衝液（pH10.5) を用いて25℃で測定した。
【０１１７】
以下の表では、親酵素（配列番号１）及び問題の変異体において、温度依存的な活性、並びに、37℃での活性に対する25℃での活性の比率％を示している。

pH7.3 に合わせた50mM Britton Robinson 緩衝液による各変異体溶液を用いて、前記のPhadebas検査によってもう１つの測定を行った。試料中の活性を、50mM Britton Robinson 緩衝液(pH7.3) を用いて37℃及び50℃で測定した。
【０１１８】
以下の表では、親酵素（配列番号１）及び問題の変異体において、温度依存的な活性、並びに、50℃での活性に対する37℃での活性の比率％を示している。

Ｂ．配列番号２の配列の変異体におけるαアミラーゼ活性
pH7.3 に合わせた50mM Britton Robinson 緩衝液による各変異体溶液を用いて、前記のPhadebas検査によって測定を行った。試料中の活性を、50mM Britton Robinson 緩衝液(pH7.3) を用いて25℃及び37℃で測定した。
【０１１９】
以下の表では、親酵素（配列番号２）及び問題の変異体において、温度依存的な活性、並びに、37℃での活性に対する25℃での活性の比率％を示している。

Ｃ．配列番号４の配列の変異体におけるαアミラーゼ活性
pH7.3 に合わせた50mM Britton Robinson 緩衝液による各変異体溶液を用いて、前記のPhadebas検査によって測定を行った。試料中の活性を、50mM Britton Robinson 緩衝液(pH7.3) を用いて37℃で、そして50mM CAPS 緩衝液（pH10.5) を用いて60℃で測定した。
【０１２０】
以下の表では、親酵素（配列番号４）及び問題の変異体において、温度依存的な活性、並びに、60℃での活性に対する37℃での活性の比率％を示している。

実施例10
BAN:1-300/Termamyl:301-483 から成る親のハイブリッドαアミラーゼに由来する変異体の構築
プラスミドpTVB191 は、BAN:1-300/Termamyl:301-483からなるハイブリッドαアミラーゼをコードする遺伝子と共に、バチルス・ズブチリス内で機能する複製起点、及びクロラムフェニコール耐性を付与するcat 遺伝子を含んでいる。
【０１２１】
変異体BM4 (F290E) を、プラスミドpTVB191 を鋳型としてメガプライマー法（Sarkar and Sommer, 1990)によって構築した。
プライマーｐ１（配列番号52）及び変異生成オリゴヌクレオチドbm4 （配列番号47）を用いて標準条件下でPCR を行って、 444bp断片を増幅した。この断片をアガロースゲルから精製して、これをメガプライマーとして、プライマーｐ２（配列番号53）と共に用いて２回目のPCR を行い、 531bp断片を得た。この断片を制限エンドヌクレアーゼHindIII 及びTth111I によって切断した。これによって生じた 389bp断片を、同酵素で切断したプラスミドpTVB191 内に連結した。できたプラスミドによってバチルス・ズブチリスSHA273を形質転換した。クロラムフェニコール及び不溶性でんぷんを含有したプレート上で増殖させることによって、クロラムフェニコール耐性クローンを選択した。そのプレートをヨウ素蒸気で染色した後、染色輪を形成したクローンを選択することによって、活性αアミラーゼを発現するクローンを単離した。DNA 配列の決定によって、導入された変異の存在を確認した。
【０１２２】
変異体BM5 (F290K), BM6 (F290A), BM8 (Q360E) 及びBM11 (N102D)を同様にして構築した。それらの構築の詳細を以下に記す。
変異体：BM5 (F290K)
変異生成オリゴヌクレオチド：bm5 （配列番号48）
プライマー（１回目 PCR) ：ｐ１（配列番号52）
生成断片サイズ： 444bp
プライマー（２回目 PCR) ：ｐ２（配列番号53）
制限エンドヌクレアーゼ：HinDIII, Tth111I
切断断片サイズ： 389bp

【０１２３】
変異体：BM6 (F290A)
変異生成オリゴヌクレオチド：bm6 （配列番号49）
プライマー（１回目 PCR) ：ｐ１（配列番号52）
生成断片サイズ： 444bp
プライマー（２回目 PCR) ：ｐ２（配列番号53）
制限エンドヌクレアーゼ：HinDIII, Tth111I
切断断片サイズ： 389bp

【０１２４】
変異体：BM8 (Q360E)
変異生成オリゴヌクレオチド：bm8 （配列番号50）
プライマー（１回目 PCR) ：ｐ１（配列番号52）
生成断片サイズ： 230bp
プライマー（２回目 PCR) ：ｐ２（配列番号53）
制限エンドヌクレアーゼ：HinDIII, Tth111I
切断断片サイズ： 389bp

【０１２５】
変異体：BM11 (N102D)
変異生成オリゴヌクレオチド：bm11（配列番号51）
プライマー（１回目 PCR) ：ｐ３（配列番号54）
生成断片サイズ： 577
プライマー（２回目 PCR) ：ｐ４（配列番号55）
制限エンドヌクレアーゼ：HinDIII, PvuI
切断断片サイズ： 576
変異生成オリゴヌクレオチド
bm4 （配列番号47）：F290E
プライマー 5' GTG TTT GAC GTC CCG CTT CAT GAG AAT TTA CAG G
bm5 （配列番号48）：F290K
プライマー 5' GTG TTT GAC GTC CCG CTT CAT AAG AAT TTA CAG G
bm6 （配列番号49）：F290A
プライマー 5' GTG TTT GAC GTC CCG CTT CAT GCC AAT TTA CAG G
bm8 （配列番号50）：Q360E
プライマー 5' AGG GAA TCC GGA TAC CCT GAG GTT TTC TAC GG
bm11（配列番号51）：N102D
プライマー 5' GAT GTG GTT TTG GAT CAT AAG GCC GGC GCT GAT G
その他のプライマー
ｐ１： 5' CTG TTA TTA ATG CCG CCA AAC C （配列番号52）
ｐ２： 5' G GAA AAG AAA TGT TTA CGG TTG CG（配列番号53）
ｐ３： 5' G AAA TGA AGC GGA ACA TCA AAC ACG （配列番号54）
ｐ４： 5' GTA TGA TTT AGG AGA ATT CC (配列番号55）
実施例11
BAN:1-300/Termamyl:301-483 から成る親のハイブリッドαアミラーゼに由来する変異体におけるアルカリ pH でのαアミラーゼ活性
各酵素溶液を用いて、Phadebas検査（前記）によって測定を行った。NaOHによって指示したpHに合わせた50mM Britton-Robinson 緩衝液中で30℃にて15分間インキュベーションした後に、活性を測定した。
NU／mg酵素
pH wt Q360E F290A F290K F290E N102D
8.0 5300 7800 8300 4200 6600 6200
9.0 1600 2700 3400 2100 1900 1900
【０１２６】
引用文献
Klein, C., et al., Biochemistry 1992, 31, 8740-8746,
Mizuno, H., et al., J. Mol. Biol. (1993) 234, 1282-1283,
Chang, C., et al, J. Mol. Biol. (1993) 229, 235-238,
Larson, S.B., J. Mol. Biol. (1994) 235, 1560-1584,
Lawson, C.L., J. Mol. Biol. (1994) 236, 590-600,
Qian, M., et al., J. Mol. Biol. (1993) 231, 785-799,
Brady, R.L., et al., Acta Crystallogr. sect. B, 47, 527-535,
Swift, H.J., et al., Acta Crystallogr. sect. B, 47, 535-544
A. Kadziola, Ph.D. Thesis:“An alpha-amylase from Barley and its Complex with a Substrate Analogue Inhibitor Studied by X-ray Crystallography ”, Department of Chemistry University of Copenhagen 1993
MacGregor, E.A., Food Hydrocolloids, 1987, Vol.1, No. 5-6, p. B. Diderichsen and L. Christiansen, Cloning of a maltogenic α-amylase from Bacillus stearothermophilus, FEMS Microbiol. letters: 56: pp. 53-60 (1988)
Hudson et al., Practical Immunology, Third edition (1989), Blackwell Scientific Publications,
Sambrook et al., Molecular Cloning : A Laboratory Manual, 2nd Ed., Cold Spring Harbor, 1989
S.L. Beaucage and M.H. Caruthers, Tetrahedron Letters 22, 1981, pp. 1859-1869
Matthes et al., The EMBO J. 3, 1984, pp. 801-805.
R.K. Saiki et al., Science 239, 1988, pp. 487-491.
Morinaga et al., (1984, Biotechnology 2 : 646-639)
Nelson and Long, Analytical Biochemistry 180, 1989, pp. 147-151
Hunkapiller et al., 1984, Nature 310 : 105-111
R. Higuchi, B. Krummel, and R.K. Saiki (1988). A general method of in vitro preparation and specific mutagenesis of DNA fragments: study of protein and DNA interactions. Nucl. Acids Res. 16 : 7351-7367.
Dubnau et al., 1971, J. Mol. Biol. 56, pp. 209-221.
Gryczan et al., 1978, J. Bacteriol. 134, pp. 318-329.
S.D. Erlich, 1977, Proc. Natl. Acad. Sci. 74, pp. 1680-1682.
Boel et al., 1990, Biochemistry 29, pp. 6244-6249.

【図面の簡単な説明】
【図１】 ６つの親のTermamyl様αアミラーゼのアミノ酸配列の整列を示す。左端の数字は、以下の通り、各アミノ酸配列を示す：
１：配列番号２
２：Kaoamyl
３：配列番号１
４：配列番号５
５：配列番号４
６：配列番号３
【図２】 SP722 （配列番号２）（pH９における）、及びB. licheniformisαアミラーゼ（配列番号４）（pH7.3 における）の温度活性曲線を示す。
【図３】 SP690 （配列番号１）、SP722 （配列番号２）、及びB. licheniformisαアミラーゼ（配列番号４）の、pH10における温度活性曲線を示す。
【図４】 ５つのαアミラーゼのアミノ酸配列の整列を示す。左端の数字は、以下の通り、各アミノ酸配列を示す：
１：マウスαアミラーゼ（amyp mouse）
２：ラットαアミラーゼ（amyp rat）
３：ブタ膵臓αアミラーゼ（PPA)（amyp pig）
４：ヒトαアミラーゼ（amyp human）
５：A. haloplanctis αアミラーゼ（AHA)（amy altha)
【配列表】

[0001]
(Field of the Invention)
The present invention relates to a variant having Termamyl-like α-amylase as a parent enzyme and having higher activity at medium temperature and / or high pH.
BACKGROUND OF THE INVENTION
α-Amylase (α-1,4-glucan-4-glucanohydrolase, EC3.2.1.1) is a hydrolysis agent for starch and linear and branched 1,4-glucoside oligosaccharides and polysaccharides. It consists of a group of enzymes that catalyze.
[0002]
There are numerous patent and scientific literature on this industrially very important group of enzymes. Some α-amylases, for example variants of Termamyl-like α-amylase, are described, for example, in WO90 / 11352, WO95 / 10603, WO95 / 26397, WO96 / 23873 and WO96 / 23874.
In recent literature on α-amylase, WO96 / 23874 reports data on the three-dimensional X-ray crystal structure analysis of Termamyl-like α-amylase. This α-amylase is composed of an N-terminal 300 amino acid residue of B. amyloliquefaciens α-amylase (BAN®) and a C-terminal amino acid 301-483 of B. licheniformis α-amylase (commercial product Termamyl®). And thus industrially important Bacillus α-amylase (which is included in the text in the term “Termamyl-like α-amylase”, in particular B. licheniformis, B. amyloliquefaciens (BAN) and B. stearothermophilus (BSG ( (Including registered α) amylase). Further, WO96 / 23874 describes a methodology for designing a Termamyl-like α-amylase variant with altered properties compared to the parent enzyme, based on the structural analysis of the parent Termamyl-like α-amylase.
BRIEF DESCRIPTION OF THE INVENTION
The present invention relates to α-amylose-degrading mutants derived from Termamyl-like α-amylase with improved activity at high pH and medium temperature (compared to the parent enzyme).
[0003]
The term “medium temperature” means herein 10-60 ° C., preferably 20-50 ° C., in particular 30-40 ° C.
The term “high pH” means the alkaline pH used today in washing, in particular about 8 to 10.5.
In the present specification, “low temperature α-amylase” means an α-amylase that exhibits a relatively optimal activity in a temperature range of 0 to 30 ° C.
[0004]
In the present specification, the “medium temperature α-amylase” means an α-amylase that exhibits optimal activity in a temperature range of 30 to 60 ° C. For example, SP 690 and SP 722 α-amylases are each “medium-temperature α-amylase”.
In the present specification, the “high temperature α-amylase” means an α-amylase that exhibits optimal activity in a temperature range of 60 to 110 ° C. For example, Termamyl is a “high temperature α-amylase”.
[0005]
The property changes that can be achieved in the variants of the invention are changes with respect to the following properties: enzyme stability at pH 8 to 10.5 and / or Ca at pH 8 to 10.5.²⁺Stability and / or specific activity at 10-60 ° C, preferably 20-50 ° C, in particular 30-40 ° C.
It should be noted that the relative optimum temperature often depends on the particular pH used. That is, for example, the relative optimum temperature determined at pH 8 may be substantially different from that determined at pH 10.
Effect of temperature on enzyme activity
The dynamics at and around the active site depend on temperature and amino acid composition and are very important for the relative optimum temperature of the enzyme. By comparing the kinetics of medium temperature α amylase and high temperature α amylase, one can determine areas that are important for the function of high temperature α amylase at medium temperature. The temperature activity curves of SP722 α-amylase (SEQ ID NO: 2) and B. licheniformis α-amylase (commercial product Termamyl, Novo Nordisk) (SEQ ID NO: 4) are shown in FIG.
[0006]
At the relative optimum temperature of SP722, that is, in the middle temperature range (30-60 ° C.), its absolute activity is higher than that of the homologous enzyme B. licheniformis α-amylase. The latter shows optimal activity at about 60-100 ° C. This curve depends mainly on temperature stability and the dynamics of the active site residues and their surroundings. Furthermore, this activity curve depends on the pH used and the pKa of the active site residue.
[0007]
The first point of the present invention is a mutant having Termamyl-like α-amylase as a parent and having α-amylase activity, and includes one or more mutations corresponding to the following mutations in the amino acid sequence of SEQ ID NO: 2. For variants: T141, K142, F143, D144, F145, P146, G147, R148, G149, Q174, R181, G182, D183, G184, K185, A186, W189, S193, N195, H107, K108, G109, D166, W167, D168, Q169, S170, R171, Q172, F173, F267, W268, K269, N270, D271, L272, G273, A274, L275, K311, E346, K385, G456, N457, K458, P459, G460, T461, V462, T463.
[0008]
Variants of the invention have one or more of the following substitutions or deletions:
T141A, D, R, N, C, E, Q, G, H, I, L, K, M, F, P, S, W, Y, V;
K142A, D, R, N, C, E, Q, G, H, I, L, M, F, P, S, T, W, Y, V;
F143A, D, R, N, C, E, Q, G, H, I, L, K, M, P, S, T, W, Y, V;
D144A, R, N, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, V;
F145A, D, R, N, C, E, Q, G, H, I, L, K, M, P, S, T, W, Y, V;
P146A, D, R, N, C, E, Q, G, H, I, L, K, M, F, S, T, W, Y, V;
G147A, D, R, N, C, E, Q, H, I, L, K, M, F, P, S, T, W, Y, V;
R148A, D, N, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, V;
G149A, D, R, N, C, E, Q, H, I, L, K, M, F, P, S, T, W, Y, V;
R181^* , A, D, N, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, V;
G182^* , A, D, R, N, C, E, Q, H, I, L, K, M, F, P, S, T, W, Y, V;
D183^* , A, R, N, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, V;
G184^* , A, R, D, N, C, E, Q, H, I, L, K, M, F, P, S, T, W, Y, V;
K185A, D, R, N, C, E, Q, G, H, I, L, M, F, P, S, T, W, Y, V;
A186D, R, N, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, V;
W189A, D, R, N, C, E, Q, G, H, I, L, K, M, F, P, S, T, Y, V;
S193A, D, R, N, C, E, Q, G, H, I, L, K, M, F, P, T, W, Y, V;
N195A, D, R, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, V;
H107A, D, R, N, C, E, Q, G, I, L, K, M, F, P, S, T, W, Y, V;
K108A, D, R, N, C, E, Q, G, H, I, L, M, F, P, S, T, W, Y, V;
G109A, D, R, N, C, E, Q, H, I, L, K, M, F, P, S, T, W, Y, V;
D166A, R, N, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, V;
W167A, D, R, N, C, E, Q, G, H, I, L, K, M, F, P, S, T, Y, V;
D168A, R, N, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, V;
Q169A, D, R, N, C, E, G, H, I, L, K, M, F, P, S, T, W, Y, V;
S170A, D, R, N, C, E, Q, G, H, I, L, K, M, F, P, T, W, Y, V;
R171A, D, N, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, V;
Q172A, D, R, N, C, E, G, H, I, L, K, M, F, P, S, T, W, Y, V;
F173A, D, R, N, C, E, Q, G, H, I, L, K, M, P, S, T, W, Y, V;
Q174^* , A, D, R, N, C, E, G, H, I, L, K, M, F, P, S, T, W, Y, V;
F267A, D, R, N, C, E, Q, G, H, I, L, K, M, P, S, T, W, Y, V;
W268A, D, R, N, C, E, Q, G, H, I, L, K, M, F, P, S, T, Y, V;
K269A, D, R, N, C, E, Q, G, H, I, L, M, F, P, S, T, W, Y, V;
N270A, D, R, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, V;
D271A, R, N, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, V;
L272A, D, R, N, C, E, Q, G, H, I, K, M, F, P, S, T, W, Y, V;
G273A, D, R, N, C, E, Q, H, I, L, K, M, F, P, S, T, W, Y, V;
A274D, R, N, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, V;
L275A, D, R, N, C, E, Q, G, H, I, K, M, F, P, S, T, W, Y, V;
K311A, D, R, N, C, E, Q, G, H, I, L, M, F, P, S, T, W, Y, V;
E346A, D, R, N, C, Q, G, H, I, K, L, M, F, P, S, T, W, Y, V;
K385A, D, R, N, C, E, Q, G, H, I, L, M, F, P, S, T, W, Y, V;
G456A, D, R, N, C, E, Q, H, I, L, K, M, F, P, S, T, W, Y, V;
N457A, D, R, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, V;
K458A, D, R, N, C, E, Q, G, H, I, L, M, F, P, S, T, W, Y, V;
P459A, D, R, N, C, E, Q, G, H, I, L, K, M, F, S, T, W, Y, V;
G460A, D, R, N, C, E, Q, H, I, L, K, M, F, P, S, T, W, Y, V;
T461A, D, R, N, C, E, Q, G, H, I, L, K, M, F, P, S, W, Y, V;
V462A, D, R, N, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y;
T463A, D, R, N, C, E, Q, G, H, I, L, K, M, F, P, S, W, Y, V.
[0009]
Variants having one or more of the following substitutions or deletions are preferred: K142R; S193P; N195F; K269R, Q; N270Y, R, D; K311R; E346Q; K385R; K458R; P459T; T461P;^* ; R181Q, N, S; G182T, S, N; D183^* ; G184^* ; K185A, R, D, C, E, Q, G, H, I, L, M, N, F, P, S, T, W, Y, V; A186T, S, N, I, V, R 189T, S, N, Q.
Particularly preferred are variants having deletions at positions D183 and G184, and also one or more of the following substitutions or deletions: K142R; S193P; N195F; K269R, Q; N270Y, R, D; K311R; E346Q; K385R ; K458R; P459T; T461P; Q174^* ; R181Q, N, S; G182T, S, N; K185A, R, D, C, E, Q, G, H, I, L, M, N, F, P, S, T, W, Y, V A186T, S, N, I, V, R; W189T, S, N, Q.
[0010]
Said variants of the invention have modifications that exhibit at least one of the following properties compared to the parent α-amylase:
i) improved pH stability at pH 8 to 10.5; and / or
ii) Ca at pH 8 to 10.5²⁺Improved stability; and / or
iii) Increase in specific activity at temperatures of 10-60 ° C., preferably 20-50 ° C., in particular 30-40 ° C.
Further details will be described below.
[0011]
In addition, the present invention provides a DNA construct encoding a variant of the invention; a process for preparing the variant of the invention; and various industrial products or methods, eg, in the melting of detergents or starches, alone or in combination with other enzymes Relates to the use of the variants of the invention.
The final point of the present invention relates to a method for providing an alpha amylase with an optimal pH change and / or an optimal temperature change and / or improved stability.
Nomenclature
In the present specification and claims, the usual one-letter code and three-letter code for amino acid residues are used. For ease of reference, the alpha amylase variants of the invention are named and described according to the following rules: Original amino acid: Position: Substituted amino acid.
[0012]
According to this nomenclature, for example, substitution of alanine at position 30 with asparagine is represented as Ala30Asn or A30N, and deletion of alanine at the same position is represented by Ala30^* Or A30^* And the insertion of the additional amino acid is represented as Ala30AlaLys or A30AK.
Deletion of consecutive amino acids, eg deletion of amino acids 30-33 is (30-33)^* Alternatively, it is expressed as Δ (A30-N33).
[0013]
If a particular α-amylase has a “deletion” compared to other α-amylases and there is an insertion at that position, for example aspartic acid is inserted at position 36, ^*36Asp or ^*It is expressed as 36D.
Multiple mutations are separated by +, for example, mutations in which alanine and glutamic acid are substituted with asparagine and serine at positions 30 and 34, respectively, are represented as Ala30Asp + Glu34Ser or A30N + E34S.
[0014]
When one or more amino acids are interchangeably inserted at a position, it is represented as A30N, E or A30N or A30E.
Where a suitable position for a modification is indicated without the modification being specified, it indicates that the amino acid at that position can be replaced with any amino acid. Thus, if the modification of alanine at position 30 is described without being specified, the alanine is deleted or any other amino acid, ie, R, N, D, A, C, Q , E, G, H, I, L, K, M, F, P, S, T, W, Y, V can be substituted.
Detailed Description of the Invention
Termamyl Α-amylase
It is known that α-amylase produced by Bacillus species has high homology at the amino acid level. For example, B. licheniformis α-amylase comprising the amino acid sequence of SEQ ID NO: 4 (commercial product Termamyl) is approximately 89% homologous to B. amyloliquefacien α-amylase comprising the amino acid sequence of SEQ ID NO: 5, About 79% homologous to B. stearothermophilus α-amylase comprising 3 amino acid sequences. Other homologous α-amylases include those derived from Bacillus sp. Strains NCIB 12289, NCIB 12512, NCIB 12513 or DSM 9375, all of which are described in detail in WO95 / 26397, and Tsukamoto α amylase (see SEQ ID NO: 6) described in et al., Biochemical and Biophysical Research Communications, 151 (1988), pp. 25-31 is included.
[0015]
In addition, homologous α-amylases include α-amylases produced by B. licheniformis strains (ATCC 27811) described in EP 0252666 and α-amylases described in WO91 / 00353 and WO94 / 18314. . Other commercially available Termamyl-like B. licheniformis alpha amylases include the products Optitherm (registered trademark) and Takatherm (registered trademark) (manufactured by Solvay), Maxamyl (registered trademark) (manufactured by Gist-brocades / Genencor), Spezym AA (registered trademark) and Spezyme Delta AA (registered trademark) (manufactured by Genencor) and Keistase (registered trademark) (manufactured by Daiwa) are included.
[0016]
Since these α-amylases are substantially homologous, they are considered to belong to the same group of α-amylases, that is, the “Termamyl-like α-amylase” group.
Thus, as used herein, the term “Termamyl-like α-amylase” means an α-amylase that is substantially homologous to Termamyl, ie, B. licheniformis α-amylase having the amino acid sequence of SEQ ID NO: 4, at the amino acid level. That is, the amino acid sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7 or 8 or the amino acid sequence of SEQ ID NO: 1 or 2 described in WO95 / 26397 (SEQ ID NO: 7 or 8 of this specification, respectively) All of the α-amylases having the amino acid sequence described in Tsukamoto et al., 1988 (equal to the amino acid sequence of SEQ ID NO: 6 in the present specification) are referred to as “Termamyl-like α-amylase”. Conceivable. Other Termamyl-like α-amylases are
i) at least 60%, such as at least 70%, such as at least 75%, or at least 80%, such as at least 85%, at least 90% of any of the amino acid sequences of SEQ ID NOs 1-8. % Or at least 95% homology and / or
ii) immunocrossreacts with antibodies made against at least one of the α-amylases and / or
iii) SEQ ID NO: 9, 10, 11 or 12 (DNA sequences encoding the amino acid sequences of SEQ ID NO: 1, 2, 3, 4 and 5 respectively), SEQ ID NO: 4 (stop codon TAA and WOA / 26397) In addition, a DNA sequence equivalent to the DNA sequence of SEQ ID NO: 13 of the present specification and encoding the amino acid sequence of SEQ ID NO: 8 of the present specification), and SEQ ID NO: 5 of WO95 / 26397 (SEQ ID NO: 14 of the present specification) Encoded by a DNA sequence that hybridizes to the DNA sequence encoding the particular α-amylase that is apparent from
α-amylase.
[0017]
In relation to the characteristics of i), “homology” can be determined using any conventional algorithm, preferably the GAP program in the GCG package, version 7.3 (June 1993), in which case the GAP penalty It can be determined at default values, namely GAP creation penalty 3.0 and GAP extension penalty 0.1 (Genetic Computer Group (1991) Program Manual for the GCG Package, version, 7, 575 Science Drive, Madison, Wisconsin, USA 53711).
[0018]
The structural alignment between Termamyl (SEQ ID NO: 4) and Termamyl-like α-amylase can identify equivalent / corresponding positions in other Termamyl-like α-amylases. One way to obtain this structural alignment is to use the Pile Up program in the GCG package with default gap penalty values, ie, the gap creation penalty 3.0 and the gap extension penalty 0.1. Other structural alignment methods include hydrophobic cluster analysis (Gaboriaud et al., (1987), FEBS LETTERS 224, pp. 149-155) and reverse threading (Huber, T; Torda, AE, PROTEIN SCIENCE Vol. 7 , No. 1, No. 1 pp. 142-149 (1998).
[0019]
The characteristics ii) of the α amylase, i.e. immune cross-reactivity, can be examined using an antibody raised against at least one epitope of the relevant Termamyl-like α amylase, or an antibody that reacts with that epitope. Both monoclonal or polyclonal antibodies can be made by existing methods in the art, such as those described in Hudson et al., Practical Immunology, Third edition (1989), Blackwell Scientific Publications. Immune cross-reactivity can be determined by existing tests in the art, such as Western blotting or radial immunodiffusion as described in Hudson et al., 1989. In this way, immune cross-reactivity was observed between α-amylases having the amino acid sequences of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7 or 8.
[0020]
Oligonucleotide probes used in assessing Termamyl-like α-amylase according to property iii) can be suitably prepared based on the complete or partial nucleotide or amino acid sequence of the α-amylase in question.
Appropriate conditions for test hybridization consist of pre-soak in 5 × SSC; 20% formamide, 5 × Denhardt's solution, 50 mM sodium phosphate, pH 6.8, and 50 mg denatured sonicated calf thymus DNA Prehybridization at 40 ° C. for 1 hour in solution; then hybridization at ˜40 ° C. for 18 hours in the same solution supplemented with 100 mM ATP; then in 2 × SSC, 0.2% SDS at 40 ° C. (low string ), Preferably at 50 ° C. (medium stringency), more preferably at 65 ° C. (high stringency), even more preferably at ~ 75 ° C. (ultra high stringency), 3 times for 30 minutes. Includes filter cleaning. Details regarding hybridization methods are described in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor, 1989.
[0021]
As used herein, “derived from” is encoded by the DNA sequence isolated from the strain as well as the alpha amylase produced or produced by the organism in question. It is meant to include alpha amylase and also alpha amylase produced by a host cell transformed with the DNA sequence. Finally, the term is meant to include an alpha amylase encoded by a DNA sequence of synthetic and / or cDNA origin and having the identified properties of the alpha amylase of interest. The term also refers to a mutation resulting from a modification of one or more amino acid residues of a naturally occurring α-amylase, ie, one or more amino acid residues of a naturally occurring α-amylase. It also means that it can be a body.
Parental hybrid alpha amylase
The parent α-amylase (ie, backbone α-amylase) may be a hybrid α-amylase, ie, an α-amylase comprising a combination of partial amino acid sequences derived from at least two α-amylases.
[0022]
The parent hybrid α-amylase may be one that can be determined to belong to the Termamyl-like α-amylase group based on amino acid homology and / or immune cross-reactivity and / or DNA hybridization (as described above). In this case, the hybrid α-amylase is typically derived from at least one portion of a Termamyl-like α-amylase and a termamyl-like α-amylase or non-Termamyl-like α-amylase of microbial (bacterial or fungal) and / or mammalian origin. One or more other α-amylase moieties selected.
[0023]
Thus, the parent hybrid α-amylase is a combination of at least two partial amino acids derived from Termamyl-like α-amylase, a portion derived from at least one Termamyl-like α-amylase and at least one non-Termamyl-like bacterial α-amylase. A combination of amino acids, or a combination of partial amino acids derived from at least one Termamyl-like α-amylase and at least one fungal α-amylase. The Termamyl-like α-amylase from which this partial amino acid is derived may be, for example, any of the specific Termamyl-like α-amylases referred to herein.
[0024]
For example, the parent α-amylase can comprise the C-terminus of an α-amylase derived from a strain of B. licheniformis and the N-terminal portion of an α-amylase derived from a strain of B. amyloliquefaciens or B. stearothermophilus. For example, the parent α-amylase may comprise at least 430 amino acids of the C-terminal portion of B. licheniformis α-amylase, eg, a) N-terminal 37 amino acid residues of B. amyloliquefaciens α-amylase having the amino acid sequence of SEQ ID NO: 5 And an amino acid portion corresponding to the C-terminal 445 amino acid residue of B. licheniformis α-amylase having the amino acid sequence of SEQ ID NO: 4, or the hybrid Termamyl-like α-amylase is matured The sequence of Termamyl, ie, SEQ ID NO: 4, except that the N-terminal 35 amino acid residues of the protein are replaced by BAN (mature protein), ie, the N-terminal 33 residues of B. amyloliquefaciens α-amylase of SEQ ID NO: 5. Or B) having the amino acid sequence of SEQ ID NO: 3 Including an amino acid portion corresponding to the N-terminal 68 amino acid residues of B. stearothermophilus α-amylase and an amino acid portion corresponding to the C-terminal 415 amino acid residues of B. licheniformis α-amylase having the amino acid sequence of SEQ ID NO: 4 It's okay.
[0025]
Another suitable parental hybrid α-amylase is described in WO96 / 23874 (Novo Nordisk), which is the N-terminus of BAN (B. amyloliquefaciens α-amylase) (amino acids 1 to 300 of the mature protein) and the C-terminus of Termamyl ( The amino acids 301-483 of the mature protein). Replacing one or more of the following positions of this hybrid α-amylase (BAN: 1-300 / Termamyl: 301-483) increased its activity: Q360, F290 and N102. Particularly interesting substitutions are one or more of the following substitutions: Q36E, D; F290A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T N102D, E.
[0026]
The corresponding positions on SP722 α-amylase shown in SEQ ID NO: 2 are S365, Y295 and N106. A particularly interesting corresponding substitution in the α-amylase of SEQ ID NO: 2 is one or more of the following substitutions: S365D, E; Y295 A, C, D, E, G, H, I, K, L, M, N , P, Q, R, S, T; and N106D, E.
The corresponding positions on the SP690 α-amylase shown in SEQ ID NO: 1 are S365, Y295, N106. A particularly interesting corresponding substitution of this is one or more of the following substitutions: S365D, E; Y295 A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T; N106D, E.
[0027]
The non-Termamyl-like α-amylase is, for example, a fungal α-amylase, a mammalian or plant α-amylase, or a bacterial α-amylase (different from a Termamyl-like α-amylase). Specific examples of such alpha amylases include Aspergillus oryzae TAKA alpha amylase A. niger acid alpha amylase, Bacillus subtilis alpha amylase, porcine pancreatic alpha amylase and barley alpha amylase. All of these α-amylases have been elucidated to have a structure that is significantly different from that of the typical Termamyl-like α-amylase referred to herein.
[0028]
Said fungal alpha amylases, ie those derived from A. niger and A. oryzae, are highly homologous at the amino acid level and are generally considered to belong to the same alpha amylase group. A fungal α-amylase derived from A. oryzae is commercially available under the trade name Fungamyl (registered trademark).
In addition, specific variants of the Termamyl-like α-amylase (variants of the present invention) can be modified in the usual manner in specific amino acid residues in the amino acid sequence of the specific Termamyl-like α-amylase (eg, deletion or substitution). Is referred to as other Termamyl-like α-amylase variants modified at the corresponding positions are also included in the invention (corresponding positions are the best conceivable between amino acid sequences). Determined based on amino acid sequence alignment).
[0029]
In a preferred embodiment of the invention, the α-amylase backbone is derived from B. licheniformis (as the parent Termamyl-like α-amylase) and has one of the above reference examples, eg the amino acid sequence of SEQ ID NO: 4. B. licheniformis α-amylase.
Altered properties of the variants of the invention
The relationship between the mutations present in the mutants of the present invention and changes in properties attributed thereto (changes to the parent Termamyl-like α-amylase) are discussed below.
pH 8 ~ 10.5 Improved stability
In the present invention, important mutations (including amino acid substitutions) with respect to gaining improved stability at high pH (ie pH 8-10.5) include one or more in SP722 α-amylase (having the amino acid sequence of SEQ ID NO: 2). The following mutations are included: T141, K142, F143, D144, F145, P146, G147, R148, G149, R181, A186, S193, N195, K269, N270, K311, K458, P459, T461.
[0030]
This variant of the invention has one or more of the following substitutions (according to the amino acid number of SEQ ID NO: 2):
T141A, D, R, N, C, E, Q, G, H, I, L, K, M, F, P, S, W, Y, V;
K142A, D, R, N, C, E, Q, G, H, I, L, M, F, P, S, T, W, Y, V;
F143A, D, R, N, C, E, Q, G, H, I, L, K, M, P, S, T, W, Y, V;
D144A, R, N, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, V;
F145A, D, R, N, C, E, Q, G, H, I, L, K, M, P, S, T, W, Y, V;
P146A, D, R, N, C, E, Q, G, H, I, L, K, M, F, S, T, W, Y, V;
G147A, D, R, N, C, E, Q, H, I, L, K, M, F, P, S, T, W, Y, V;
R148A, D, N, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, V;
G149A, D, R, N, C, E, Q, H, I, L, K, M, F, P, S, T, W, Y, V;
K181A, D, R, N, C, E, Q, G, H, I, L, M, F, P, S, T, W, Y, V;
A186D, R, N, C, E, Q, G, H, I, L, P, K, M, F, S, T, W, Y, V;
S193A, D, R, N, C, E, Q, G, H, I, L, K, M, F, P, T, W, Y, V;
N195A, D, R, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, V;
K269A, D, R, N, C, E, Q, G, H, I, L, M, F, P, S, T, W, Y, V;
N270A, D, R, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, V;
K311A, D, R, N, C, E, Q, G, H, I, L, M, F, P, S, T, W, Y, V;
K458A, D, R, N, C, E, Q, G, H, I, L, M, F, P, S, T, W, Y, V;
P459A, D, R, N, C, E, Q, G, H, I, L, K, M, F, S, T, W, Y, V;
T461A, D, R, N, C, E, Q, G, H, I, L, K, M, F, P, S, W, Y, V.
[0031]
Preferred high pH stable variants include those having one or more of the following substitutions in SP722 α-amylase (amino acid sequence of SEQ ID NO: 2): K142R, R181S, A186T, S193P, N195F, K269R, N270Y, K311R , K458R, P459T and T461P.
In a particular embodiment, the Bacillus strain NCIB 12512 α-amylase having the sequence of SEQ ID NO: 1, or the B. stearothermophilus α-amylase having the sequence of SEQ ID NO: 3, or the B. licheniformis α-amylase having the sequence of SEQ ID NO: 4, Alternatively, B. amyloliquefaciens α-amylase having the sequence of SEQ ID NO: 5 is used for the modification as the backbone, ie the parent Termamyl-like α-amylase.
[0032]
As can be seen from the alignment in FIG. 1, in B. stearothermophilus α-amylase, the position corresponding to N270 of SP722 is already tyrosine. Furthermore, in the Bacillus strain NCIB 12512 α-amylase, B. stearothermophilus α-amylase, B. licheniformis α-amylase and B. amyloliquefaciens α-amylase, the position corresponding to K458 of SP722 is already arginine. Furthermore, in B. licheniformis α-amylase, the position corresponding to T461 of SP722 is already proline. Therefore, these α-amylases are not suitable for substitution.
[0033]
Α-amylase variants with improved stability at high pH can be made by substituting in the regions found by molecular dynamics simulations described in Example 2. This simulation shows a region with higher flexibility or mobility at high pH (pH 8-10.5) compared to medium pH.
By utilizing the structure of any bacterial α-amylase homologous to Termamyl-like α-amylase (BA2) having the 3D structure disclosed in the attached table of WO96 / 23874 (Novo Nordisk) (see below), such α-amylase It is possible to model the structure of and to perform molecular dynamics simulations on it. The homology of the bacterial α-amylase may be at least 60%, preferably more than 70%, more preferably more than 80%, most preferably more than 90% to the Termamyl-like α-amylase (BA2). However, this is measured using the UWGCG GAP program in the GCG package, version 7.3 (June 1993) with default GAP penalty values (Genetic Computer Group (1991) Program Manual for the GCG Package, version 7, 575 Science Drive, Madison, Wisconsin, USA 53711).
[0034]
Substitution of an undesirable residue with another residue may also be applicable.
pH 8 ~ 10.5 In Ca ²⁺ Improved stability
Ca²⁺The improvement of stability is Ca²⁺It means that the enzyme stability at the time of removal is improved. In the present invention, Ca at high pH²⁺Mutations important for obtaining improved stability (including amino acid substitutions) include mutations or deletions at one or more positions corresponding to the following positions in the SP722 α-amylase having the amino acid sequence of SEQ ID NO: 2. Included: R181, G182, D183, G184, K185, A186, W189, N195, N270, E346, K385, K458, P459.
[0035]
This variant of the invention has one or more of the following substitutions or deletions:
R181^* , A, D, N, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, V;
G182^* , A, D, R, N, C, E, Q, H, I, L, K, M, F, P, S, T, W, Y, V;
D183^* , A, R, N, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, V;
G184^* , A, R, D, N, C, E, Q, H, I, L, K, M, F, P, S, T, W, Y, V;
K185A, D, R, N, C, E, Q, G, H, I, L, M, F, P, S, T, W, Y, V;
A186D, R, N, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, V;
W189A, D, R, N, C, E, Q, G, H, I, L, K, M, F, P, S, T, Y, V;
N195A, D, R, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, V;
N270A, R, D, N, C, E, Q, H, I, L, K, M, F, P, S, T, W, Y, V;
E346A, R, D, N, C, Q, G, H, I, L, K, M, F, P, S, T, W, Y, V;
K385A, R, D, N, C, E, Q, G, H, I, L, M, F, P, S, T, W, Y, V;
K458A, R, D, N, C, E, Q, G, H, I, L, M, F, P, S, T, W, Y, V;
P459A, R, D, N, C, E, Q, G, H, I, L, K, M, F, S, T, W, Y, V.
[0036]
Preferred are variants having one or more of the following substitutions or deletions: R181Q, N; G182T, S, N; D183^* ; G184^* K185A, R, D, C, E, Q, G, H, I, L, M, N, F, P, S, T, W, Y, V; A186T, S, N, I, V; W189T , S, N, Q; N195F; N270R, D; E346Q; K385R; K458R; P459T.
In a particular embodiment, a Bacillus strain NCIB 12512 α-amylase having the sequence of SEQ ID NO: 1, or B. amyloliquefaciens α-amylase having the sequence of SEQ ID NO: 5, or B. licheniformis α-amylase having the sequence of SEQ ID NO: 4 Use as a backbone for.
[0037]
As can be seen from the alignment in FIG. 1, the B. licheniformis α-amylase lacks positions corresponding to D183 and G184 of SP722. Therefore, these substitutions are not appropriate in this α-amylase.
In a preferred embodiment, the variant is an α-amylase of Bacillus strain NCIB 12512 having a deletion of D183 and G184 and one further substitution: R181Q, N and / or G182T, S, N and / or D183^* ; G184^* And / or K185A, R, D, C, E, Q, G, H, I, L, M, N, F, P, S, T, W, Y, V and / or A186T, S, N, I , V and / or W189T, S, N, Q and / or N195F and / or N270R, D and / or E346Q and / or K385R and / or K458R and / or P459T.
Increase in specific activity at medium temperature
As another aspect of the present invention, an important mutation related to obtaining a mutant having an increased specific activity at a temperature of 10 to 60 ° C., preferably 20 to 50 ° C., particularly 30 to 40 ° C. Mutations corresponding to one or more of the following positions of SP722 α-amylase having the amino acid sequence of: H107, K108, G109, D166, W167, D168, Q169, S170, R171, Q172, F173, Q174, D183, G184, N195, F267, W268, K269, N270, D271, L272, G273, A274, L275, G456, N457, K458, P459, G460, T461, V462, T463.
[0038]
This variant of the invention has one or more of the following substitutions:
H107A, D, R, N, C, E, Q, G, I, L, K, M, F, P, S, T, W, Y, V;
K108A, D, R, N, C, E, Q, G, H, I, L, M, F, P, S, T, W, Y, V;
G109A, D, R, N, C, E, Q, H, I, L, K, M, F, P, S, T, W, Y, V;
D166A, R, N, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, V;
W167A, D, R, N, C, E, Q, G, H, I, L, K, M, F, P, S, T, Y, V;
D168A, R, N, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, V;
Q169A, D, R, N, C, E, G, H, I, L, K, M, F, P, S, T, W, Y, V;
S170A, D, R, N, C, E, Q, G, H, I, L, K, M, F, P, T, W, Y, V;
R171A, D, N, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, V;
Q172A, D, R, N, C, E, G, H, I, L, K, M, F, P, S, T, W, Y, V;
F173A, D, R, N, C, E, Q, G, H, I, L, K, M, P, S, T, W, Y, V;
Q174^* , A, D, R, N, C, E, G, H, I, L, K, M, F, P, S, T, W, Y, V;
D183^* , A, D, R, N, C, E, Q, G, H, I, L, K, M, F, P, S, W, Y, V;
G184^* , A, R, N, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, V;
N195A, D, R, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, V;
F267A, D, R, N, C, E, Q, G, H, I, L, K, M, P, S, T, W, Y, V;
W268A, D, R, N, C, E, Q, G, H, I, L, K, M, F, P, S, T, Y, V;
K269A, D, R, N, C, E, Q, G, H, I, L, M, F, P, S, T, W, Y, V;
N270A, D, R, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, V;
D271A, R, N, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, V;
L272A, D, R, N, C, E, Q, G, H, I, K, M, F, P, S, T, W, Y, V;
G273A, D, R, N, C, E, Q, H, I, L, K, M, F, P, S, T, W, Y, V;
A274D, R, N, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, V;
L275A, D, R, N, C, E, Q, G, H, I, K, M, F, P, S, T, W, Y, V;
G456A, D, R, N, C, E, Q, H, I, L, K, M, F, P, S, T, W, Y, V;
N457A, D, R, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, V;
K458A, D, R, N, C, E, Q, G, H, I, L, M, F, P, S, T, W, Y, V;
P459A, D, R, N, C, E, Q, G, H, I, L, K, M, F, S, T, W, Y, V;
G460A, D, R, N, C, E, Q, H, I, L, K, M, F, P, S, T, W, Y, V;
T461A, D, R, N, C, E, Q, G, H, I, L, K, M, F, P, S, W, Y, V;
V462A, D, R, N, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y;
T463A, D, R, N, C, E, Q, G, H, I, L, K, M, F, P, S, W, Y, V.
[0039]
Preferred variants have one or more of the following substitutions or deletions: Q174^* , D183^* , G184^* , K269S.
In a particular embodiment, B. licheniformis α-amylase having the sequence of SEQ ID NO: 4 is used as the backbone of these mutations.
General mutations in the variants of the invention: increased specific activity at moderate temperatures
Particularly interesting amino acid substitutions are those that increase the mobility around the active site of the enzyme. This is accomplished by changes that break stable interactions in the vicinity of the active site, ie, from any residue that constitutes the active site, preferably within 10 Å or 8 Å or 6 Å or 4 Å.
[0040]
Examples of this mutation include mutations that reduce side chain size, such as:
Ala to Gly
From Val to Ala or Gly
From Ile or Leu to Val, Ala or Gly
From Thr to Ser.
[0041]
Such mutations are expected to increase the flexibility of the active site region by introduction of cavities or structural restructuring to fill the space left by the mutation.
The mutations of the present invention may preferably include one or more modifications in addition to the mutations outlined above. Thus, advantageously, the one or more proline residues present in that portion of the alpha amylase variant to be modified are any possible non-proline residue in nature, preferably alanine, glycine, serine, threonine, It can be replaced by valine or leucine.
[0042]
Similarly, preferably one or more cysteine residues present in the amino acid residue of the parent α-amylase to be modified are replaced by non-cysteine residues such as serine, alanine, threonine, glycine, valine or leucine. Can be done.
Further, in the variant of the present invention, one or more Asp and / or Glu present in the amino acid fragment corresponding to amino acid fragment 185 to 209 of SEQ ID NO: 4 are each independently by Asu and / or Gln, or It can be substituted with any of the modifications outlined above. In addition, in Termamyl-like α-amylase, substitution of one or more Lys residues present in the amino acid fragment corresponding to amino acid fragments 185 to 209 of SEQ ID NO: 4 with Arg is also noted.
[0043]
The present invention is intended to include variants that incorporate two or more of the above outlined modifications.
Furthermore, it may be advantageous to introduce point mutations in any of the variants described herein.
Alpha amylase mutant with increased motility around the active site
The motility of the alpha amylase variants of the invention can be increased by substituting one or more amino acids at one or more positions near the substrate site. Such positions are V56, K108, D168, Q169, Q172, L201, K269, L272, L275, K446, P459 (according to the number of SP722 α-amylase of SEQ ID NO: 2).
[0044]
Accordingly, one aspect of the present invention relates to a variant having a mutation at one or more of said positions.
Preferred substitutions are one or more of the following substitutions:
V56A, G, S, T;
K108A, D, E, Q, G, H, I, L, M, N, S, T, V;
D168A, G, I, V, N, S, T;
Q169A, D, G, H, I, L, M, N, S, T, V;
Q172A, D, G, H, I, L, M, N, S, T, V;
L201A, G, I, V, S, T;
K269A, D, E, Q, G, H, I, L, M, N, S, T, V;
L272A, G, I, V, S, T;
L275A, G, I, V, S, T;
Y295A, D, E, Q, G, H, I, L, M, N, F, S, T, V;
K446A, D, E, Q, G, H, I, L, M, N, S, T, V;
P459A, G, I, L, S, T, V.
[0045]
In particular embodiments, an α-amylase of Bacillus strain NCIB 12512 having the sequence of SEQ ID NO: 1, a B. stearothermophilus α-amylase having the sequence of SEQ ID NO: 3, a B. licheniformis α-amylase having the sequence of SEQ ID NO: 4, or of SEQ ID NO: 5 B. amyloliquefaciens α-amylase having the sequence is used as the backbone of these mutations.
[0046]
As can be seen from the alignment in FIG. 1, B. licheniformis α-amylase and B. amyloliquefaciens α-amylase have glutamine at a position corresponding to K269 of SP722. Furthermore, B. stearothermophilus α-amylase has a serine at a position corresponding to K269 of SP722. Therefore, in these α-amylases, these substitutions are not appropriate.
[0047]
Furthermore, as can be seen from the alignment in FIG. 1, B. amyloliquefaciens α-amylase has an alanine at a position corresponding to L272 of SP722, and B. stearothermophilus α-amylase has an isoleucine at a position corresponding to L272 of SP722. Therefore, these α-amylases are not suitable for substitution.
As can be seen from the alignment in FIG. 1, the α-amylase of Bacillus strain 12512 has an isoleucine at a position corresponding to L722 of SP722. Therefore, the substitution is not appropriate in this α-amylase.
[0048]
As can be seen from the alignment in FIG. 1, B. amyloliquefaciens α-amylase has a phenylalanine at a position corresponding to Y295 of SP722. Furthermore, B. stearothermophilus α-amylase has asparagine at a position corresponding to Y295 of SP722. Therefore, these α-amylases are not suitable for substitution.
As can be seen from the alignment in FIG. 1, B. licheniformis α-amylase and B. amyloliquefaciens α-amylase have asparagine at a position corresponding to K446 of SP722. Furthermore, B. stearothermophilus α-amylase has histidine at a position corresponding to K446 of SP722. Therefore, these α-amylases are not suitable for substitution.
[0049]
As can be seen from the alignment in FIG. 1, B. licheniformis α-amylase, B. amyloliquefaciens α-amylase, and B. stearothermophilus α-amylase have serine at a position corresponding to P459 of SP722. Furthermore, the α-amylase of Bacillus strain NCIB 12512 has a threonine at a position corresponding to P459 of SP722. Therefore, these α-amylases are not suitable for substitution.
Stabilization of enzymes with high activity at moderate temperatures
In another aspect, the present invention relates to a low temperature alpha amylase (such as that of Asteromonas haloplanctis (Feller et al., (1994), Eur. J. Biochem. 222: 441-447)), and mesophilic alpha having activity at moderate temperatures. It relates to improving the stability of amylases (eg SP722 and SP690), ie the enzymes commonly known as psychrophilic and mesophilic enzymes. Stability in this particular group of enzymes can be interpreted as temperature stability or stability under calcium deficiency conditions.
[0050]
Typically, enzymes that exhibit high activity at moderate temperatures expose serious problems in conditions that stress the enzyme, such as temperature or calcium deficiency.
The object of the present invention is therefore to provide an enzyme which exhibits the desired high activity at moderate temperatures without losing its activity under slightly loaded conditions.
The activity of this stabilizing variant measured at medium temperature is preferably more than 100% to 50%, more preferably more than 100% to 70% of the original activity at a specific temperature before enzyme stabilization, and most preferably Preferably it should be above 100% to 85%. The produced enzyme must be able to withstand a longer incubation under stressed conditions than the wild-type enzyme.
[0051]
Possible enzymes include, for example, alpha amylases from bacteria or fungi.
An example of such a low temperature alpha amylase is that isolated from Alteromonas haloplanctis (Feller et al., (1994), Eur. J. Biochem. 222: 441-447). The crystal structure of this α-amylase has been elucidated (Aghajari et al., (1988), Protein Science 7: 564-572).
[0052]
This A. haloplanctis α-amylase (AHA) (No. 5 in the alignment of FIG. 4) has about 66% homology to porcine pancreatic α-amylase (PPA) (No. 3 in the alignment of FIG. 4). Show. The 3D structure of PPA is known and can be obtained from the Brookhaven database under the name 1OSE or 1DHK. Based on homology to other more stable α-amylases, stabilization of the “cold high activity enzyme” derived from Alteromonas haloplanctis α amylase can be obtained simultaneously with maintaining the desired high activity at moderate temperatures.
[0053]
FIG. 4 shows the sequence alignment of five α-amylases, including AHA and PPA α-amylases. Specific mutations that improve the stability of Alteromonas haloplanctis alpha amylase are T66P, Q69P, R155P, Q177R, A205P, A232P, L243R, V295P, S315R.
Method for preparing α-amylase mutant
Several methods for introducing mutations into genes are known in the art. After briefly examining the cloning of the DNA sequence encoding α-amylase, a method for generating a mutation at a specific site in the coding sequence of α-amylase will be described.
Encodes α-amylase DNA Sequence cloning
The DNA sequence encoding the parent α-amylase can be isolated from any cell or microorganism producing the α-amylase by a variety of methods well known in the art. First, a genomic DNA and / or cDNA library must be constructed using chromosomal DNA or messenger RNA derived from the organism that produces the α-amylase being studied. Next, if the amino acid sequence of the alpha amylase is known, a homologous labeled oligonucleotide probe is synthesized and used to clone alpha amylase from a genomic library prepared from the organism in question. Can be identified. Alternatively, clones encoding α-amylase can be identified by hybridization and washing under lower stringency conditions using a labeled oligonucleotide probe having a sequence homologous to a known α-amylase gene.
[0054]
Yet another method of identifying clones encoding α-amylase is to insert a fragment of genomic DNA into an expression vector, eg, a plasmid, transform the α-amylase negative bacteria with the resulting genomic DNA library, and Agar plates containing the substrate are seeded with transformed bacteria, thereby obtaining a clone that expresses the alpha amylase to be identified.
[0055]
Alternatively, the DNA sequence encoding the enzyme can be obtained by established standard methods, such as the phosphoramidite method described in SL Beaucage and MH Caruthers (1981) or the method described in Matthes et al. (1984). It may also be prepared synthetically. In the phosphoramidite method, oligonucleotides are synthesized by, for example, an automatic DNA synthesizer, purified, annealed, ligated into an appropriate vector, and cloned.
[0056]
Finally, the DNA sequence may be a genomic-derived and synthetic-derived mixture, a synthetic-derived and cDNA-derived mixture, or a genomic-derived and cDNA-derived mixture, which is a synthetic-derived, genomic-derived or cDNA-derived fragment. (As appropriate fragments corresponding to various regions of the complete DNA sequence) can be prepared by ligation according to standard methods. The DNA sequence may be prepared by polymerase chain reaction (PCR) using specific primers, for example as described in US 4,683,202 or R.K. Saiki et al. (1988).
Expression of α-amylase mutant
In the present invention, DNA sequences encoding variants made by the above methods or alternative methods known in the art are typically represented by promoters, operators, ribosome binding sites, translation initiation signals, and optionally repressors. It can be expressed in the form of an enzyme using an expression vector containing the gene or various activator genes.
[0057]
The recombinant expression vector carrying the DNA sequence encoding the α-amylase variant of the present invention may be any vector that can be conveniently subjected to DNA recombination technology. Often depends on the host cell Thus, the vector can be an autonomously replicating vector, ie a vector that is entirely extrachromosomal and replicates independently of chromosomal replication, such as a plasmid, bacteriophage or extrachromosomal element, minichromosome or artificial chromosome. Alternatively, the vector may be a vector that, when introduced into a host cell, integrates into the host cell genome and replicates with the integrated chromosome.
[0058]
Within the vector, the DNA sequence must be operably linked to an appropriate promoter sequence. The promoter is any DNA sequence that exhibits transcriptional activity in the selected host cell and may be obtained from a gene encoding a protein that is homologous or heterologous to the host cell. Examples of promoters suitable for directing transcription of a DNA sequence encoding an α-amylase variant of the present invention are those that exhibit transcriptional activity in a selected host cell and are homologous or heterologous to that host cell. A sequence that can be obtained from the encoding gene. Examples of promoters suitable for directing transcription of a DNA sequence encoding an α-amylase variant of the invention, particularly in bacterial hosts, include the E. coli lac operon, the Streptomyces coelicolor agarase gene dagA promoter, Bacillus licheniformis α-amylase Gene (amyL) promoter, Bacillus stearothermophilus maltose amylase gene (amyM) promoter, Bacillus amyloliquefaciens α-amylase (amyQ) promoter, Bacillus subtilis xylA and xylB gene promoters. Examples of promoters useful for transcription in fungal hosts are A. oryzae TAKA amylase, Rhizomucor miehei aspartate proteinase, A. niger neutral α-amylase, A. niger acid-stable α-amylase, A. niger glucoamylase, Rhizomucor miehei It is derived from a gene encoding lipase, A. oryzae alkaline protease, A. oryzae triose phosphate isomerase, or A. nidulans acetamidase.
[0059]
The expression vector of the present invention may comprise a suitable transcription terminator, and in the case of eukaryotes, a polyadenylation sequence, which is operably linked to a DNA sequence encoding an alpha amylase variant of the present invention. . This stop sequence and polyadenylation sequence can be suitably obtained from the same source as the promoter.
The vector may further comprise a DNA sequence that enables the vector to replicate in the host cell in question. Examples of such sequences are the origins of replication in plasmids pUC19, pACYC177, pUB110, pE194, pAMB1 and pIJ702.
[0060]
The vector further comprises a selectable marker, for example a gene for a product that complements the defect in the host cell, such as the dal gene from B. subtilis or B. licheniformis, or ampicillin, kanamycin, chloramphenicol or tetracycline. A gene that confers resistance to antibiotics. In addition, the vector may contain selectable markers of Aspergillus, such as amdS, argB, niaD and sC, ie markers that confer hygromycin resistance, or may be co- (simultaneous) as described, for example, in WO91 / 17243 Selection may be performed via transformation.
[0061]
In some respects, for example, when using bacteria as host cells, intracellular expression may be advantageous, but generally extracellular expression is preferred. In general, the Bacillus α-amylase described herein includes a pre-region that causes the expressed protease to be secreted into the medium. If desired, this front region may be replaced by a different front region or signal sequence. This is conveniently accomplished by replacement of the DNA sequence encoding each front region.
[0062]
A method for ligating each of the DNA construct of the present invention encoding an α-amylase variant, a promoter, a terminator, and other elements, and a method for inserting them into an appropriate vector containing information necessary for replication are as follows. Well known to those skilled in the art (eg, Sambrook et al., Molecular Cloning: A Laboraotry Manual, 2nd Ed., Cold Spring Harbor, 1989).
[0063]
Advantageously, the cells of the invention containing either the DNA constructs or expression vectors of the invention as described above are used as host cells in the recombinant production of the α-amylase variants of the invention. This cell can be transformed with the DNA construct of the invention encoding the variant. This is conveniently done by integrating the DNA construct (with a copy number of 1 or more) into the host chromosome. This integration will generally be advantageous because the DNA sequence is more likely to be stably maintained in the cell. Integration of the DNA construct into the host chromosome can be performed according to conventional methods, for example by homologous or non-homologous recombination. Alternatively, the cells may be transformed with the expression vectors in different types of host cells.
[0064]
Examples of suitable bacteria are gram-positive bacteria such as Bacillus subtilis, Bacillus licheniformis, Bacillus lentus, Bacillus brevis, Bacillus stearothermophilus, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus coagulans, Bacillus circulans, Bacillus megaterus, Bacillus lividans or Streptomyces murinus, or Gram-negative bacteria such as E. coli. Bacterial transformation can be performed according to known methods, for example using protoplasts or competent cells.
[0065]
The yeast may preferably be selected from Saccharomyces or Schizosaccharomyces strains such as Saccharomyces cerevisiae. The filamentous fungus may advantageously be an Aspergillus species such as A. oryzae or A. niger. Fungal transformation can be performed according to known methods in a manner comprising protoplast formation, protoplast transformation, and cell wall regeneration. A suitable method for transformation of Aspergillus host cells is described in EP 238023.
[0066]
In yet another aspect, the present invention relates to a method of producing an α-amylase variant of the invention, the method comprising culturing the host cell under conditions that induce production of the variant, and the cell and Recovering the variant from the medium.
The medium in which the cells are cultured can be any conventional medium suitable for growing the host cells in question and expressing the alpha amylase variant of the invention. Appropriate media can be purchased from commercial suppliers or prepared according to reported compositions (eg, those described in catalogs of the American Type Culture Collection).
[0067]
The α-amylase variant secreted from the host cell can be easily recovered from the medium by a well-known method, including separation of the cell from the medium by centrifugation or filtration, medium with a salt such as ammonium sulfate, etc. Precipitation of medium protein components and the use of chromatography such as ion exchange chromatography or affinity chromatography are included.
Industrial availability
The α-amylase variants of the present invention have valuable properties for various uses in industry. In particular, the enzyme variant of the present invention can be used as a component in cleaning compositions for cleaning, dishwashing and hard surface cleaning.
[0068]
A number of variants are particularly useful for sweetener and ethanol production from starch and / or desizing of textiles. Conditions for normal starch conversion, such as starch liquefaction and / or saccharification, are described, for example, in US Pat. No. 3,912,590 and EP Patent Publications 252,730 and 63,909.
Cleaning composition
As described above, the variants of the present invention can be suitably incorporated into a cleaning composition. With respect to suitable components in cleaning compositions (eg laundry or dishwashing detergents), suitable methods of incorporating the variants into such cleaning compositions, and with examples of suitable types of cleaning compositions, see eg WO96 See / 23874 and WO97 / 07202.
[0069]
The cleaning composition containing the variants of the present invention may further comprise one or more other enzymes, such as lipases, cutinases, proteases, cellulases, peroxidases or laccases, and / or other alpha amylases.
The alpha amylase variant of the present invention can be incorporated into the cleaning composition to a concentration that is normally used. It is presently believed that the variants of the invention can be incorporated in amounts corresponding to 0.00001-1 mg (as pure active enzyme protein) per liter of washing / dishwashing liquid, according to the normal addition level of detergent.
[0070]
The present invention also relates to a method for providing an α-amylase, 1) the optimum pH has changed and / or 2) the optimum temperature has changed and / or 3) improved stability. Comprises the following process:
i) by comparing the molecular dynamics of the 3D structure of two or more alpha amylases having substantially different properties of pH, temperature and / or stability, thereby targeting the location and / or region for that alpha amylase mutation Identifying;
ii) substitution, addition and / or deletion of one or more amino acids in their identified position and / or region.
[0071]
In an embodiment of the invention, the medium temperature alpha amylase is compared to the high temperature alpha amylase. In another embodiment, the low temperature alpha amylase is compared to either a medium temperature or high temperature alpha amylase.
The α-amylases to be compared should preferably be at least 70%, preferably 80%, up to 90%, for example up to 95%, in particular 95% homologous to each other.
[0072]
The α-amylase to be compared may be Termamyl-like α-amylase as described above. In certain embodiments, the alpha amylase to be compared is the alpha amylase set forth in SEQ ID NOs: 1-8.
In another aspect, the stability characteristics of the alpha amylase in question are Ca²⁺It is a dependent characteristic.
[Materials and methods]
enzyme
SP722 (SEQ ID NO: 2, available from Novo Nordisk)
Termamyl (SEQ ID NO: 4, available from Novo Nordisk)
SP690 (SEQ ID NO: 1, available for purchase from Novo Nordisk)
Bacillus subtilis SHA273: See WO95 / 10603
Plasmid
pJE1 contains a gene encoding a variant of SP722 α-amylase, ie, a variant lacking 6 nucleotides corresponding to amino acids D183-G184 of the mature protein. Transcription of the JE1 gene is controlled by the amyL promoter. This plasmid further contains an origin of replication derived from the plasmid pUB110 (Gryczan, TJ et al. (1978) J. Bact. 134: 318-329) and a cat gene conferring resistance to kanamycin.
<Method>
Library vector pDorK101 Building
The E. coli / Bacillus shuttle vector pDorK101 (below) can be used to introduce mutations without expressing α-amylase in E. coli and to modify α-amylase to be active in Bacillus. This vector was constructed as follows. In pJE1, a JE1 coding gene (SP722 lacking D183-G184) was obtained by interrupting the gene at the PstI site in the 5 ′ coding region of SEQ ID NO: 2 with a 1.2 kb fragment containing the E. coli replication origin. Inactivated. The fragment was amplified by PCR from pUC19 (GenBank Access No. X02514) using a forward primer: 5'-gacctgcagtcaggcaacta-3 'and a reverse primer: 5'-tagagtcgacctgcaggcat-3'. The PCR amplified fragment and pJE1 vector were digested with PstI at 37 ° C. for 2 hours. The pJE1 vector fragment and the PCR fragment were ligated at room temperature for 1 hour, whereby E. coli was transformed by an electrical method. The resulting vector is called pDorK101.
Filter screening test
Using this test method, a Termamyl-like α-amylase mutant with improved stability at high pH compared to the parent enzyme, and stable at higher pH and medium temperature compared to the parent enzyme, depending on the set temperature at the time of screening. Termamyl-like α-amylase variants with improved properties can be screened.
High pH Filter inspection
The Bacillus library can be obtained from cellulose acetate filters (OE 67, Schleicher & Schuell, Dassel, Germany) and nitrocellulose filters (Protran-Ba 85, Schleicher & Schuell, Dassel, Germany) on TY agar plates containing 10 μg / ml kanamycin. ) And incubate at 37 ° C for at least 21 hours. Place the cellulose acetate layer on the TY agar plate.
[0073]
After plating, each sandwich filter is marked with a needle before incubation to position the positive mutant on the filter. The mutant-bound nitrocellulose filter is transferred to a container containing glycine / NaOH buffer (pH 8.6 to 10.6) and incubated for 15 minutes at room temperature (variable in the range 10-60 ° C). Store the cellulose acetate filter with bound colonies on a TY plate at room temperature until use. After incubation, the remaining activity is detected on a plate comprising 1% agarose, 0.2% starch and glycine / NaOH buffer (pH 8.6 to 10.6). The test plate with the nitrocellulose filter is marked and incubated for 2 hours at room temperature, as in the case of the sandwich filter. After removing the filter, the test plate is stained with 10% Lugol solution. Variants that degrade starch are detected as white spots on a dark blue background and identified on storage plates. Positive mutants are rescreened twice under the same conditions as the first screening method.
Low calcium filter inspection
The Bacillus library can be obtained using cellulose acetate filters (OE 67, Schleicher & Schuell, Dassel, Germany) and nitrocellulose filters (Protran-Ba 85) on TY agar plates containing appropriate antibiotics such as kanamycin or chloramphenicol. , Schleicher & Schuell, Dassel, Germany) and incubate at 37 ° C for at least 21 hours. Place the cellulose acetate layer on the TY agar plate.
[0074]
After plating, each sandwich filter is marked with a needle before incubation to position the positive mutant on the filter. The mutant-bound nitrocellulose filter is transferred to a container containing carbonate / bicarbonate buffer (pH 8.5-10) and different concentrations of EDTA (0.001 mM to 100 mM). The filter is incubated for 1 hour at room temperature. Store the cellulose acetate filter with bound colonies on a TY plate at room temperature until use. After incubation, the remaining activity is detected on a plate comprising 1% agarose, 0.2% starch and carbonate / bicarbonate buffer (pH 8.5-10). The test plate with the nitrocellulose filter is marked and incubated for 2 hours at room temperature, as in the case of the sandwich filter. After removing the filter, the test plate is stained with 10% Lugol solution. Variants that degrade starch are detected as white spots on a dark blue background and identified on storage plates. Positive mutants are rescreened twice under the same conditions as the first screening method.
How to get the area of interest
The 3D structures of three bacterial α-amylases are known. Among them, two B. licheniformis alpha amylases, 1BPL from Brookhaven database (Machius et al. (1995), J. Mol. Biol. 246, p. 545-559) and 1VJS (Song et al. (1996), Enzymes for Carbohydrate 163 Engineering (Prog. Biotechnol. V 12)). In these two structures, important fragments in the structure are deleted from the so-called B domain in the region surrounding the binding site of two calcium ions and one sodium ion. Therefore, the present inventor used the 3D structure of α-amylase BA2 (WO96 / 23874; hybrid of BAN (SEQ ID NO: 5) and B. licheniformis α-amylase (SEQ ID NO: 4)). Based on the structure, models of B. licheniformis α-amylase and SP722 α-amylase were constructed.
Fermentation and purification of α-amylase mutants
Fermentation and purification can be performed by methods well known in the art.
Determination of stability
All stability tests are performed with the same settings. The method is shown below. The enzyme is incubated under appropriate conditions (1-4). At various time points, for example after 0, 5, 10, 15 and 30 minutes, samples are taken and diluted 25-fold in test buffer (0.1 M 50 mM Britton buffer pH 7.3). Activity is measured by Phadebas test (Pharmacia) under standard conditions of pH 7.3, 37 ° C.
[0075]
The activity measured before incubation (at 0 min) is used as a reference value (100%). The percent reduction is calculated as a function of incubation time. In the table, for example, the residual activity after 30 minutes of incubation is shown.
Determination of specific activity
Specific activity is determined as activity / mg enzyme using the Phadebas test (Pharmacia). Follow the manufacturer's instructions (see “Testing α-amylase activity” below).
α-Amylase activity test
1. Phadebas inspection
Α-amylase activity is determined by the method using Phadebas® tablets as a substrate. Phadebas tablets (Phadebas Amylase Test, Pharmacia Diagnostic) are tablets containing cross-linked insoluble blue starch polymer mixed with bovine serum albumin and buffer.
[0076]
For each measurement, 5 tablets of 50 mM Britton-Robinson buffer (50 mM acetic acid, 50 mM phosphoric acid, 50 mM boric acid, 0.1 mM CaCl₂ , And adjust the pH to the set point with NaOH). The test is performed in a water bath at the set temperature. The alpha amylase to be tested is diluted in xml 50 mM Britton-Robinson buffer. 1 ml of this alpha amylase solution is added to 5 ml of 50 mM Britton-Robinson buffer. Starch is hydrolyzed by α-amylase to produce a soluble blue fragment. The absorbance of the resulting blue solution measured with a spectrophotometer at 620 nm is a function of α-amylase activity.
[0077]
It is important that the absorbance at 620 nm measured after 10 or 15 minutes of incubation (inspection time) is in the range of 0.2 to 2 absorbance units. Within this range of absorbance, there is a proportional relationship between activity and absorbance (Lambert-Beer law). Therefore, it is necessary to adjust the dilution of the enzyme to meet this standard. Under certain conditions (temperature, pH, reaction time, buffer conditions), 1 mg of a α-amylase hydrolyzes a certain amount of substrate and a blue color is produced. The intensity of this color is measured at 620 nm. Under certain conditions, the measured absorbance is directly proportional to the specific activity of the α-amylase in question (activity / mg pure α-amylase protein).
2. Alternative method
Α-amylase activity is determined by the method using PNP-G7 as a substrate. PNP-G7 (abbreviation for p-nitrophenyl-α, D-maltoheptaoside) is a protected oligosaccharide that can be cleaved by endoamylase. After cleavage, α-glucosidase included in the kit digests the substrate and releases free PNP molecules. This molecule is yellow and is measured by a visible spectrophotometer with a wavelength of 405 nm (400-420 nm). A kit containing PNP-G7 substrate and α-glucosidase is manufactured and sold by Boehringer-Mannheim (Catalog No. 1054635).
[0078]
To prepare the substrate, add one bottle of substrate (BM 1442309) to 5 ml buffer (BM 1442309). To prepare α-glucosidase, one bottle of α-glucosidase (BM 1462309) is added to 45 ml buffer (BM 1442309). Mix 5 ml of this α-glucosidase solution with 0.5 ml of substrate solution to make a working solution.
For this test, 20 μl of enzyme solution is added to a 96 well microtiter plate and incubated at 25 ° C. Add 200 ml of working solution at 25 ° C. This solution is mixed and incubated for 1 minute, and then the absorbance at 405 nm is measured every 15 seconds for 3 minutes.
[0079]
Under certain conditions, the slope of the absorbance change curve is directly proportional to the specific activity (activity / mg enzyme) of the α-amylase in question.
DOPE General method of random mutagenesis using a program
Random mutagenesis can be performed according to the following process:
1. Select the region of interest for modification in the parent enzyme;
2. Determining the mutational and non-mutant sites in the selected region;
3. Determining the type of mutation to be induced, eg depending on the desired stability and / or performance of the mutant to be constructed;
4). Select structurally rational mutations;
5. Adjusting the residues selected according to step 3 according to step 4;
6). Analyze nucleotide distribution according to the appropriate dope algorithm;
7). If necessary, match the required residues to the actual genetic code (eg, taking into account constraints arising from the genetic code (eg to avoid introduction of stop codons)) Is known to those skilled in the art and cannot be used, and therefore requires adjustment);
8). Make a primer;
9. Random mutagenesis is performed using the primer;
Ten. The resulting α-amylase variant is selected by screening for the desired improvement in properties.
[0080]
Suitable dope algorithms for use in step 6 are well known in the art. One algorithm is described in Tomandl, D. et al., Journal of Computer-Aided Molecular Design, 11 (1997), pp. 29-38). Another algorithm DOPE is described below.
dope program
The “DOPE” program is a useful computer program for optimizing the nucleotide composition of a triple codon so as to encode an amino acid distribution that most closely approximates the required amino acid distribution. An evaluation function is needed to evaluate which of the possible distributions most closely approximates the required amino acid distribution. In the "DOPE" program, the following functions have been found suitable:
[0081]
[Expression 1]

[0082]
Where x_i Is the amount of amino acids and amino acid groups calculated by the program, y_i Is the required amount of amino acids and groups of amino acids as determined by the user of the program (eg, any of the usual 20 amino acids or stop codons, eg, a certain percentage (eg 90% Ala, 3% Ile, 7% Val) to determine if it is required to be introduced), and w_i Is the assigned weighting factor determined by the user of the program (eg, depending on the importance of having a particular amino acid residue inserted at the position in question). N is the number of amino acid groups determined by the user of this program +21. For this function, 0⁰ Is 1.
[0083]
In order to find the maximum of this function, the Monte-Carlo algorithm (one example is Valleau, JP & Whittington, SG (1977) A guide to Mont Carlo for statistical mechanics: 1 Highways. In “Stastistical Mechanics, Part A” Equlibrium Techniqeues ed. BJ Berne, New York: Plenum). In each iteration, the following process occurs:
1. At each base, a new random nucleotide composition is selected, in which case the absolute difference between the current composition and the new composition is such that all three positions of the codon are four nucleotides G, A, T, C Is less than or equal to d in each case (see below for definition of d).
2. The evaluation score of the new composition and the evaluation score of the composition at that time are compared using the function s. If the new score is greater than or equal to the current score, the new composition is retained and the current composition is changed to the new composition. If the new evaluation score is smaller, the probability of retaining the new composition is exp [1000 [new evaluation score−current evaluation score]].
[0084]
A cycle typically consists of 1000 iterations as described above, where d decreases linearly from 1 to 0. In optimization, 100 cycles or more are performed. The nucleotide composition with the highest score is presented last.
〔Example〕
Example 1:
Termamyl Example of homology construction
The overall homology of B. licheniformis α-amylase (hereinafter termed “Termamyl”) with other Termamyl-like α-amylases is high, and the similarity is extremely high. Termamyl similarity to BSG (B. stearothermophilus α-amylase with SEQ ID NO: 3) and BAN (B. awyloliquefaciens α-amylase with SEQ ID NO: 5) calculated using the GCG program (University of Wisconsin Genetics Computer Group's program) Were 89% and 78%, respectively. Termamyl lacks two residues between residues G180 and K181 compared to BAN and BSG. BSG lacks 3 residues between G371 and I372 compared to BAN and Termamyl. At the N-terminus, BAN has 2 fewer residues and Termamyl has 1 fewer residues than BSG.
[0085]
A model of each structure of B. licheniformis α-amylase (Termamyl) and B. awyloliquefaciens α-amylase (BAN) was constructed based on the structure disclosed in Appendix Table 1 of WO96 / 23974. The structure of other Termamyl-like α-amylases (eg, those disclosed herein) can be constructed in a similar manner.
Compared to the alpha amylase used to elucidate the structure, Termamyl differs in that two residues near 178-182 are deleted. To compensate for this in the model structure, BIOSYM's HOMOLOGY program was used to replace residues in the corresponding position of the structure (not just the structurally conserved region) except for the deletion point. A peptide bond was established between G179 (G177) and K180 (K180) in Termamyl (BAN). There are very few atoms found that the close structural relationship between the solved structure and the model structure (and hence the effectiveness of the latter) is too close to each other in the model Is pointed out.
[0086]
The INSIGHT program then converted all of the water (605) from the elucidated structure into the very coarse structure of Termamyl at the same coordinates as the elucidated structure (see Appendix 1 of WO96 / 23874). And ions (4 calcium and 1 sodium) were added. In this case, only a slight overlap was observed, ie a very good fit. This model structure was then minimized by a 200-step Steepest descent and a 600-step Conjugated gradient (Brooks et al., 1983, J. Computational Chemistry 4, p. 187- 217). Next, molecular dynamics was applied to the minimized structure, and heating was performed at 5 ps to an equilibrium of 200 ps over 35 ps. This kinetic was operated according to the Verlet algorithm and maintained an equilibrium temperature of 300 K according to the conjugation with the water environment by Behrendsen (Berendsen et al., 1984, J. Chemical Physics 81, p. 3684-3690). Rotation and translation were taken every picosecond.
Example 2:
pH A method for deriving an important region for identifying alpha-amylase variants that exhibit improved stability and temperature-dependent activity changes
Molecular dynamics simulation is performed on the X-ray analysis structure and / or model construction structure of the enzyme of interest, here SP722 and Termamyl. This molecular dynamics simulation is performed by the CHARMM (Molecular simulations (MSI)) program or other suitable program such as DISCOVER (MSI). This molecular dynamics analysis is performed on the enzyme in an isolated state, or more preferably in a state containing crystal water, or in water, for example, embedded in a water bulb or water box. This simulation is operated for a period of 300 picoseconds (ps) or more, for example, 300-1200 ps. For the Cα carbon of the structure, an isotropic vibration is taken and compared between the structures. When there is a deletion and / or insertion in one sequence, an isotropic vibration derived from the other structure is inserted, and the difference in isotropic variation is made zero. See the CHARMM instruction manual (available from MSI) for a description of isotropic vibration.
[0087]
This molecular dynamics simulation can be performed according to standard charges for charged amino acids. That is, Asp and Glu are negatively charged and Lys and Arg are positively charged. This condition approximates a neutral pH of about 7. For analysis at higher or lower pH, altered pKa of standard titratable residues can be obtained, usually in the range of pH 2-10, to titrate the molecule. Such residues are Lys, Arg, Asp, Glu, Tyr and His. Set, Thr and Cys can be titrated, but are not considered here. Here, with respect to the charge changed due to pH, at high pH Asp and Glu are negative and Arg and Lys are uncharged. This approximates a pH of about 10-11, in which case the standard pKa for Lys and Arg is about 9-11 and begins titration of these residues.
1. Methods used to derive key regions for identifying alpha amylase variants with high pH stability:
An important area for constructing mutants with improved pH stability is the area that exhibits the best motility at extreme pH, i.e., the area with the greatest isotropic oscillation.
[0088]
Such regions are identified by performing the following two molecular dynamics simulations: i) The basic amino acids Lys and Arg are neutral (ie unprotonated) and the acidic amino acids Asp and Glu Operation at a high pH with a charge (-1), and ii) a neutral pH at which the basic amino acids Lys and Arg have a net charge (+1) and the acidic amino acid has a charge (-1) Operation in.
[0089]
Comparing the two operations, a region was identified that showed a relatively higher motility at high pH compared to neutral pH.
Residues that improve general stability, such as hydrogen bonding, residues that make the region more rigid (eg, due to mutations such as proline substitution of glycine residues), or charge or interresidue interactions By introducing residues that improve, the high pH stability of the enzyme is improved.
2. Methods for extracting regions to identify alpha amylase variants with increased activity at moderate temperatures:
An important region for constructing mutants with increased activity at medium temperature is the difference in the isotropic vibration between SP722 and Termamyl, ie the difference between the isotropic vibration of Cα in SP722 minus that in Termamyl. I found out. The region where the mobility of the isotropic vibration was the maximum was selected. Such a region and its residues were expected to increase activity at moderate temperatures. If there is adequate motility at those residues, only the activity of α-amylase is expressed. If the motility of those residues is too low, the activity is reduced or disappears.
Example 3:
Local random dope Compared to the parent enzyme by mutagenesis Ca ²⁺ Improved stability Termamyl Of amyloid-like α-amylase
In order to improve the stability of alpha amylase at low calcium concentrations, random mutagenesis was performed in a preselected region.
Area: SAI
Residue: R181-W189
The DOPE program (see "Materials and Methods") was used to determine the spiked codon for each mutation suggested in the SAI region to minimize the amount of stop codons (Table 1). . To obtain a suggested group of amino acid mutations, the exact distribution of nucleotides at the three positions of the codon was calculated. In order to increase the probability of making it the desired residue, the region of interest was specifically processed at the indicated position, but there may still be other possibilities.
Table 1:
Distribution of amino acid residues at each position

Table 2 shows the resulting treated oligonucleotide strand as the sense strand, along with the wild-type nucleotide and amino acid sequences and the nucleotide distribution at each treatment position.
Table 2:

Random mutagenesis
21 bases using the spiked oligonucleotide (referred to as FSA) and reverse primer RSA for the SAI region and SP722 (SEQ ID NO: 2) specific primer from SacII to DraII sites evident from Table 2 PCR library fragments are generated by the overlap extension method by pair duplication (Horton et al., Gene 77 (1989), pp. 61-68). The template for this PCR is plasmid pJE1. This PCR fragment can be cloned into the E. coli / Bacillus shuttle vector pDorK101 (see “Materials and Methods”) so that it can be mutagenized in E. coli and immediately expressed in Bacillus subtilis. Avoid lethal accumulation of amylase. After establishing a cloned PCR fragment in E. coli, the modified pUC19 fragment is excised from the plasmid, and the promoter and the mutant Termamyl gene are naturally ligated, thus allowing expression in Bacillus. Become.
screening
This library can be screened with a low calcium filter test as described in Materials and Methods.
Example 4:
Amylase of SEQ ID NO: 1 (SP690) Construction of mutants
The gene encoding the amylase of SEQ ID NO: 1 is present in the plasmid pTVB106 described in WO96 / 23873. The amylase is expressed from the amyL promoter of this construct in Bacillus subtilis.
[0090]
One variant of the protein is delta (T183-G184) + Y243F + Q391E + K444Q. The construction of this mutant is described in WO96 / 23873.
The construction of delta (T183-G184) + N195F by the megaprimer method described in Sarkar and Sommer, (1990), BioTechniques 8: 404-407 is shown below.
PTVB106-like plasmid (having a delta (T183-G184) mutation in the gene encoding amylase of SEQ ID NO: 1) using gene-specific primer B1 (SEQ ID NO: 17) and mutagenesis primer 101458 (SEQ ID NO: 19) Thus, a DNA fragment of about 645 bp was amplified by PCR.
[0091]
This 645 bp fragment was purified from an agarose gel, and this was used as a megaprimer together with primer Y2 (SEQ ID NO: 18), and a second PCR was performed on the same template.
The resulting approximately 1080 bp fragment was cleaved with restriction enzymes BstEII and AflIII, the resulting approximately 510 bp DNA fragment was purified, and a pTVB106-like plasmid digested with the same enzyme (delta in the gene encoding amylase of SEQ ID NO: 1). (T183-G184) having a mutation). This ligation transforms competent Bacillus subtilis SHA273 cells, and the chloramphenicol resistant transformants are examined by DNA sequencing to confirm that the appropriate mutations are present on the plasmid. did.
Primer B1: (SEQ ID NO: 17)
5 'CGA TTG CTG ACG CTG TTA TTT GCG 3'
Primer Y2: (SEQ ID NO: 18)
5 'CTT GTT CCC TTG TCA GAA CCA ATG 3'
Primer 101458: (SEQ ID NO: 19):
5 'GT CAT AGT TGC CGA AAT CTG TAT CGA CTT C 3'
Mutant delta (T183-G184) + K185R + A186T was constructed in the same manner as above except that mutagenic primer 101638 was used.
Primer 101638: (SEQ ID NO: 20)
5 'CC CAG TCC CAC GTA CGT CCC CTG AAT TTA TAT ATT TTG 3'
Mutant: Delta (T183-G184) + A186T, Delta (T183-G184) + A186I, Delta (T183-G184) + A186S, Delta (T183-G184) + A186N, pTVB106-like plasmid (mutant delta (T183-G184) + K185R + A186T) Is used as a template and a cloning vector. The mutagenic oligonucleotide (Oligo 1) for that purpose is
5 'CC CAG TCC CAG NTCTTT CCC CTG AAT TTA TAT ATT TTG 3' (SEQ ID NO: 21)
It is.
[0092]
N means that a mixture of four bases: A, C, G and T was used in the synthesis of this mutagenic oligonucleotide. The correct codon at amino acid position 186 of the mature protein is confirmed by DNA sequencing of the transformant.
Mutant delta (T183-G184) + K185R + A186T + N195F is constructed as follows.
[0093]
PCR is performed on pTVB106-like plasmid (having a mutation of delta (T183-G184) + K185R + A186T) using primer x2 (SEQ ID NO: 22) and primer 101458 (SEQ ID NO: 19). Using the resulting DNA fragment as a megaprimer and primer Y2 (SEQ ID NO: 18), PCR is performed on a pTVB106-like plasmid (having a mutation of delta (T183-G184) + N195F). This second PCR product was cut with the restriction endonucleases Acc65I and AflIII and cloned into a pTVB106-like plasmid (Delta (T183-G184) + N195F) cut with the same enzymes.
Primer x2: (SEQ ID NO: 22)
5 'GCG TGG ACA AAG TTT GAT TTT CCT G 3'
Mutant: Delta (T183-G184) + K185R + A186T + N195F + Y243F + Q391E + K444Q is constructed as follows.
[0094]
PCR is performed on pTVB106-like plasmid (having a mutation of delta (T183-G184) + K185R + A186T) using primer x2 and primer 101458. Using the resulting DNA fragment as a megaprimer and primer Y2, PCR is performed on a pTVB106-like plasmid (having a mutation of delta (T183-G184) + Y243F + Q391E + K444Q). This second round PCR product was cut with the restriction endonucleases Acc65I and AflIII and cloned into a pTVB106-like plasmid (Delta (T183-G184) + Y243F + Q391E + K444Q) cut with the same enzymes.
Example 5
Parent enzyme SP722 Construction of a site-specific α-amylase mutant in α-amylase (SEQ ID NO: 2)
A variant of amylase (SP722) of SEQ ID NO: 2 is constructed as follows.
[0095]
The gene encoding the amylase of SEQ ID NO: 2 is present in the plasmid pTVB112 described in WO96 / 23873. This amylase is expressed from the amyL promoter of this construct in Bacillus subtilis.
The construction of delta (D183-G184) + V56I by the megaprimer method described in Sarkar and Sommer, 1990 (BioTechniques 8: 404-407) is shown below.
[0096]
Using a gene-specific primer DA03 and a megaprimer DA07, a DNA fragment of about 820 bp was PCR-derived from a pTVB112-like plasmid (which has a delta (D183-G184) mutation in the gene encoding α-amylase of SEQ ID NO: 2). Amplify by.
This 820 bp fragment is purified from an agarose gel, and this is used as a megaprimer with primer DA01 to perform the second PCR against the same template.
[0097]
The resulting fragment of about 920 bp was cleaved with restriction enzymes NgoMI and AatII, and the resulting DNA fragment of about 170 bp was purified, and a pTVB112-like plasmid cleaved with the same enzyme (delta ( D183-G184) having a mutation). This ligation transforms competent Bacillus subtilis SHA273 cells (low amylase and low protease), and the chloramphenicol resistant transformants are examined by DNA sequencing to find the appropriate plasmid on the plasmid. Confirm that the mutation exists.
Primer DA01: (SEQ ID NO: 23)
5 'CCTAATGATGGGAATCACTGG 3'
Primer DA03: (SEQ ID NO: 24)
5 'GCATTGGATGCTTTTGAACAACCG 3'
Primer DA07: (SEQ ID NO: 25)
5 'CGCAAAATGATATCGGGTATGGAGCC 3'
Mutants: Delta (D183-G184) + K108L, Delta (D183-G184) + K108Q, Delta (D183-G184) + K108E, Delta (D183-G184) + K108V to Sarkar and Sommer, 1990 (BioTechniques 8: 404-407) Constructed by the described megaprimer method.
[0098]
PCR is performed on the pTVB112-like plasmid (having the mutation of delta (D183-G184)) using primer DA03 and mutagenesis primer DA20. The resulting DNA fragment is used as a megaprimer together with primer DA01 to perform PCR on a pTVB112-like plasmid (having a mutation in delta (D183-G184)). A fragment of about 920 bp generated by the second PCR is cleaved with restriction enzymes AatII and MluI and ligated to a pTVB112-like plasmid (having a delta (D183-G184) mutation) cleaved with the same enzymes.
Primer DA20: (SEQ ID NO: 26):
5 'GTGATGAACCACSWAGGTGGAGCTGATGC 3'
S means to use a mixture of two bases C and G in synthesizing the mutagenic oligonucleotide, and W means to use a mixture of two bases A and T in that case. Sequencing of the transformants confirms that an appropriate codon is present at amino acid position 108 of the mature amylase.
[0099]
Mutants: Delta (D183-G184) + D168A, Delta (D183-G184) + D168I, Delta (D183-G184) + D168V, Delta (D183-G184) + D168T were constructed in the same manner except that mutagenesis primer DA14 was used. To do.
Primer DA14 (SEQ ID NO: 27):
5 'GATGGTGTATGGRYCAATCACGACAATTCC 3'
R means to use a mixture of two bases A and G in synthesizing the mutagenic oligonucleotide, and Y means to use a mixture of two bases C and T in that case. Sequencing of the transformants confirms that an appropriate codon is present at amino acid position 168 of the mature amylase.
[0100]
Mutant: Delta (D183-G184) + Q169N is constructed in the same manner except that mutagenic primer DA15 is used.
Primer DA15 (SEQ ID NO: 28):
5 'GGTGTATGGGATAACTCACGACAATTCC 3'
Mutant: Delta (D183-G184) + Q169L is constructed in the same manner except that mutagenic primer DA16 is used.
Primer DA16 (SEQ ID NO: 29):
5 'GGTGTATGGGATCTCTCACGACAATTCC 3'
Mutant: Delta (D183-G184) + Q172N is constructed in the same manner except that the mutagenic primer DA17 is used.
Primer DA17 (SEQ ID NO: 30):
5 'GGGATCAATCACGAAATTTCCAAAATCGTATC 3'
Mutant: Delta (D183-G184) + Q172L is constructed in the same manner except that mutagenic primer DA18 is used.
Primer DA18 (SEQ ID NO: 31):
5 'GGGATCAATCACGACTCTTCCAAAATCGTATC 3'
Mutant: Delta (D183-G184) + L201I is constructed in the same manner except that the mutagenic primer DA06 is used.
Primer DA06 (SEQ ID NO: 32):
5 'GGAAATTATGATTATATCATGTATGCAGATGTAG 3'
Mutant: Delta (D183-G184) + K269S is constructed in the same way except that mutagenic primer DA09 is used.
Primer DA09 (SEQ ID NO: 33):
5 'GCTGAATTTTGGTCGAATGATTTAGGTGCC 3'
Mutant: Delta (D183-G184) + K269Q is constructed in the same manner except that the mutagenic primer DA11 is used.
Primer DA11 (SEQ ID NO: 34):
5 'GCTGAATTTTGGTCGAATGATTTAGGTGCC 3'
Mutant: Delta (D183-G184) + N270Y is constructed in the same manner except that mutagenic primer DA21 is used.
Primer DA21 (SEQ ID NO: 35):
5 'GAATTTTGGAAGTACGATTTAGGTCGG 3'
Mutants: Delta (D183-G184) + L272A, Delta (D183-G184) + L272I, Delta (D183-G184) + L272V, Delta (D183-G184) + L272T are constructed in the same manner except that the mutagenic primer DA12 is used. .
Primer DA12 (SEQ ID NO: 36):
5 'GGAAAAACGATRYCGGTGCCTTGGAGAAC 3'
R means to use a mixture of two bases A and G in synthesizing the mutagenic oligonucleotide, and Y means to use a mixture of two bases C and T in that case. Sequencing of the transformants confirms that an appropriate codon is present at amino acid position 272 of the mature amylase.
[0101]
Mutants: Delta (D183-G184) + L275A, Delta (D183-G184) + L275I, Delta (D183-G184) + L275V, Delta (D183-G184) + L275T are constructed in the same manner except that mutagenesis primer DA13 is used. .
Primer DA13 (SEQ ID NO: 37):
5 'GATTTAGGTGCCTRYCAGAACTATTTA 3'
R means to use a mixture of two bases A and G in synthesizing the mutagenic oligonucleotide, and Y means to use a mixture of two bases C and T in that case. Sequencing of the transformants confirms that an appropriate codon is present at amino acid position 275 of the mature amylase.
[0102]
Mutant: Delta (D183-G184) + Y295E is constructed in the same manner except that mutagenic primer DA08 is used.
Primer DA08 (SEQ ID NO: 38):
5 'CCCCCTTCATGAGAATCTTTATAACG 3'
The construction of delta (D183-G184) + K446Q by the megaprimer method described in Sarkar and Sommer, 1990 (BioTechniques 8: 404-407) is shown below.
[0103]
A pTVB112-like plasmid (a gene encoding amylase of SEQ ID NO: 2 having a delta (D183-G184) mutation using a gene-specific primer DA04 and a mutagenesis primer DA10 that anneals 214-231 bp downstream of the stop codon ), A DNA fragment of about 350 bp was amplified by PCR.
Using the resulting DNA fragment as a megaprimer and primer DA05, PCR is performed on a pTVB112-like plasmid (having a mutation in delta (D183-G184)). The approximately 460 bp product from this second round of PCR is cut with the restriction enzymes SnaBI and NotI and cloned into a pTVB112-like plasmid (Delta (D183-G184)) cut with the same enzymes.
Primer DA04 (SEQ ID NO: 39):
5 'GAATCCGAACCTCATTACACATTCG 3'
Primer DA05 (SEQ ID NO: 40):
5 'CGGATGGACTCGAGAAGGAAATACCACG 3'
Primer DA10 (SEQ ID NO: 41):
5 'CGTAGGGCAAAATCAGGCCGGTCAAGTTTGG 3'
Mutant: Delta (D183-G184) + K458R is constructed in the same manner except that mutagenic primer DA22 is used.
Primer DA22 (SEQ ID NO: 42):
5 'CATAACTGGAAATCGCCCGGGAACAGTTACG 3'
Mutants: Delta (D183-G184) + P459S and Delta (D183-G184) + P459T are constructed in the same manner except that mutagenic primer DA19 is used.
Primer DA19 (SEQ ID NO: 43):
5 'CTGGAAATAAAWCCGGAACAGTTACG 3'
W means using a mixture of two bases A and T when synthesizing the mutagenic primer. Sequencing of the transformants confirms that an appropriate codon is present at amino acid position 459 of the mature amylase.
[0104]
Mutant: Delta (D183-G184) + T461P is constructed in the same manner except that mutagenic primer DA23 is used.
Primer DA23 (SEQ ID NO: 44):
5 'GGAAATAAACCAGGACCCGTTACGATCAATGC 3'
Mutant: Delta (D183-G184) + K142R is constructed in the same manner except that the mutagenic primer DA32 is used.
Primer DA32 (SEQ ID NO: 45):
5 'GAGGCTTGGACTAGGTTTGATTTTCCAG 3'
Mutant: Delta (D183-G184) + K269R is constructed in the same manner except that mutagenic primer DA31 is used.
Primer DA31 (SEQ ID NO: 46):
5 'GCTGAATTTTGGCGCAATGATTTAGGTGCC 3'
Example 6
Parental Termamyl Construction of a site-specific α-amylase variant in α-amylase (SEQ ID NO: 4)
The amyL gene encoding Termamylα-amylase is present in the plasmid pDN1528 described in WO95 / 10603 (Novo Nordisk). Substitution mutants N265R and N265D each derived from this parent are constructed by the method described in WO97 / 41213 or the “megaprimer” method described above. The mutagenic primers are as follows:
Primer b11 for N265R substitution:
5 'PCC AGC GCG CCT AGG TCA CGC TGC CAA TAT TCA G (SEQ ID NO: 56)
Primer b12 for N265D substitution:
5 'PCC AGC GCG CCT AGG TCA TCC TGC CAA TAT TCA G (SEQ ID NO: 57)
P represents a phosphate group.
Example 7
Alkaline in a variant derived from a parent α-amylase having the amino acid sequence of SEQ ID NO: 2 pH In pH Determination of stability
In this series of analysis, purified enzyme samples were used. Measurement was performed using each mutant solution with 100 mM CAPS buffer adjusted to pH 10.5. This solution was incubated at 75 ° C.
[0105]
After incubation for 20 and 30 minutes, residual activity was measured by PNP-G7 test (see “Materials and Methods”). Residual activity in the sample was measured using Britton Robinson buffer (pH 7.3). The decrease in residual activity was measured compared to the 0 min time point of the corresponding reference solution of the same enzyme that was not incubated at high pH and 75 ° C.
In the table below, the parent enzyme (SEQ ID NO: 2) and the variant in question show the percentage relative to it as a function of the initial activity.
[0106]

In another series of analyses, the culture supernatant was used. Measurement was performed using each mutant solution with 100 mM CAPS buffer adjusted to pH 10.5. This solution was incubated at 80 ° C.
[0107]
After 30 minutes incubation, residual activity was measured by Phadebas test (see “Materials and Methods”). Residual activity in the sample was measured using Britton Robinson buffer (pH 7.3). The decrease in residual activity was measured relative to the 0 min time point of the corresponding reference solution of the same enzyme that was not incubated at high pH and 80 ° C.
In the table below, the parent enzyme (SEQ ID NO: 2) and the variant in question show the percentage relative to it as a function of the initial activity.
[0108]

Example 8
Alkalis in mutants derived from the parent α-amylase having the amino acid sequences of SEQ ID NOs: 1, 2 and 4 pH Of calcium stability at high temperatures
A. Calcium stability in variants of the sequence of SEQ ID NO: 1
Measurement was carried out using each mutant solution with 100 mM CAPS buffer adjusted to pH 10.5. Polyphosphoric acid was added to this solution to a final concentration of 2400 ppm (at t = 0). This solution was incubated at 50 ° C.
[0109]
After incubation for 20 and 30 minutes, residual activity was measured by PNP-G7 test (described above). Residual activity in the sample was measured using Britton Robinson buffer (pH 7.3). The decrease in residual activity was measured relative to the 0 minute time point of the corresponding reference solution of the enzyme that was not incubated at high pH and 50 ° C.
In the table below, the parent enzyme (SEQ ID NO: 1) and the mutant in question show the percentage relative to it as a function of the initial activity.
[0110]

n.d. = undecided
B. Calcium stability in variants of the sequence of SEQ ID NO: 2.
In this series of analysis, purified enzyme samples were used. Measurement was carried out using each mutant solution with 100 mM CAPS buffer adjusted to pH 10.5. Polyphosphoric acid was added to this solution to a final concentration of 2400 ppm (at t = 0). This solution was incubated at 50 ° C.
[0111]
After incubation for 20 and 30 minutes, residual activity was measured by PNP-G7 test (described above). Residual activity in the sample was measured using Britton Robinson buffer (pH 7.3). The decrease in residual activity was measured relative to the 0 minute time point of the corresponding reference solution of the enzyme that was not incubated at high pH and 50 ° C.
In the table below, the parent enzyme (SEQ ID NO: 2) and the variant in question show the percentage relative to it as a function of the initial activity.
[0112]

In another series of analyses, the culture supernatant was used. Measurement was carried out using each mutant solution with 100 mM CAPS buffer adjusted to pH 10.5. Polyphosphoric acid was added to this solution to a final concentration of 2400 ppm (at t = 0). This solution was incubated at 50 ° C.
[0113]
After incubation for 30 minutes, residual activity was measured by the Phadebas test described above. Residual activity in the sample was measured using Britton Robinson buffer (pH 7.3). The decrease in residual activity was measured relative to the 0 minute time point of the corresponding reference solution of the enzyme that was not incubated at high pH and 50 ° C.
In the table below, the parent enzyme (SEQ ID NO: 2) and the variant in question show the percentage relative to it as a function of the initial activity.
[0114]

C. Calcium stability in a variant of the sequence of SEQ ID NO: 4
Measurement was performed using each mutant solution with 100 mM CAPS buffer adjusted to pH 10.5. Polyphosphoric acid was added to this solution to a final concentration of 2400 ppm (at t = 0). This solution was incubated at 60 ° C.
[0115]
After incubation for 20 minutes, residual activity was measured by PNP-G7 test (described above). Residual activity in the sample was measured using Britton Robinson buffer (pH 7.3). The decrease in residual activity was measured relative to the 0 min time point of the corresponding reference solution of the same enzyme that was not incubated at high pH and 60 ° C.
In the table below, the parent enzyme (SEQ ID NO: 4) and the variant in question show the percentage relative to it as a function of the initial activity.
[0116]

Example 9
Measurement of activity at medium temperature in α-amylase having the amino acid sequence of SEQ ID NO: 1
A. Α-amylase activity in a variant of the sequence of SEQ ID NO: 1
Measurement was carried out by the above-mentioned Phadebas test using each mutant solution with 50 mM Britton Robinson buffer adjusted to pH 7.3. Activity in the samples was measured at 37 ° C. using 50 mM Britton Robinson buffer (pH 7.3) and at 25 ° C. using 50 mM CAPS buffer (pH 10.5).
[0117]
The table below shows the temperature-dependent activity and the ratio of the activity at 25 ° C. to the activity at 37 ° C. in the parent enzyme (SEQ ID NO: 1) and the mutant in question.

Another measurement was performed by the above-mentioned Phadebas test using each mutant solution in 50 mM Britton Robinson buffer adjusted to pH 7.3. The activity in the sample was measured at 37 ° C. and 50 ° C. using 50 mM Britton Robinson buffer (pH 7.3).
[0118]
The table below shows the temperature-dependent activity and the ratio of the activity at 37 ° C. to the activity at 50 ° C. for the parent enzyme (SEQ ID NO: 1) and the mutant in question.

B. Α-amylase activity in a variant of the sequence of SEQ ID NO: 2
Measurement was carried out by the above-mentioned Phadebas test using each mutant solution with 50 mM Britton Robinson buffer adjusted to pH 7.3. The activity in the sample was measured at 25 ° C. and 37 ° C. using 50 mM Britton Robinson buffer (pH 7.3).
[0119]
The table below shows the temperature dependent activity and the ratio of the activity at 25 ° C. to the activity at 37 ° C. in the parent enzyme (SEQ ID NO: 2) and the mutant in question.

C. Α-amylase activity in a variant of the sequence of SEQ ID NO: 4
Measurement was carried out by the above-mentioned Phadebas test using each mutant solution with 50 mM Britton Robinson buffer adjusted to pH 7.3. Activity in the samples was measured at 37 ° C. using 50 mM Britton Robinson buffer (pH 7.3) and at 60 ° C. using 50 mM CAPS buffer (pH 10.5).
[0120]
The table below shows the temperature dependent activity and the ratio of the activity at 37 ° C. to the activity at 60 ° C. for the parent enzyme (SEQ ID NO: 4) and the mutant in question.

Example 10
BAN: 1-300 / Termamyl: 301-483 Of a variant derived from a parent hybrid α-amylase consisting of
The plasmid pTVB191 contains a gene encoding a hybrid α-amylase consisting of BAN: 1-300 / Termamyl: 301-483, a replication origin that functions in Bacillus subtilis, and a cat gene conferring chloramphenicol resistance. Yes.
[0121]
Mutant BM4 (F290E) was constructed by the megaprimer method (Sarkar and Sommer, 1990) using plasmid pTVB191 as a template.
PCR was performed under standard conditions using primer p1 (SEQ ID NO: 52) and mutagenic oligonucleotide bm4 (SEQ ID NO: 47) to amplify a 444 bp fragment. This fragment was purified from an agarose gel, and a second PCR was performed using this as a megaprimer and primer p2 (SEQ ID NO: 53) to obtain a 531 bp fragment. This fragment was cleaved with restriction endonucleases HindIII and Tth111I. The resulting 389 bp fragment was ligated into plasmid pTVB191 cut with the same enzyme. Bacillus subtilis SHA273 was transformed with the resulting plasmid. Chloramphenicol resistant clones were selected by growing on plates containing chloramphenicol and insoluble starch. After the plate was stained with iodine vapor, clones expressing active α-amylase were isolated by selecting clones that formed staining wheels. The presence of the introduced mutation was confirmed by determination of the DNA sequence.
[0122]
Mutants BM5 (F290K), BM6 (F290A), BM8 (Q360E) and BM11 (N102D) were similarly constructed. Details of their construction are given below.
Mutant: BM5 (F290K)
Mutation oligonucleotide: bm5 (SEQ ID NO: 48)
Primer (first PCR): p1 (SEQ ID NO: 52)
Generated fragment size: 444bp
Primer (second PCR): p2 (SEQ ID NO: 53)
Restriction endonuclease: HinDIII, Tth111I
Cut fragment size: 389bp

[0123]
Mutant: BM6 (F290A)
Mutation oligonucleotide: bm6 (SEQ ID NO: 49)
Primer (first PCR): p1 (SEQ ID NO: 52)
Generated fragment size: 444bp
Primer (second PCR): p2 (SEQ ID NO: 53)
Restriction endonuclease: HinDIII, Tth111I
Cut fragment size: 389bp

[0124]
Mutant: BM8 (Q360E)
Mutation oligonucleotide: bm8 (SEQ ID NO: 50)
Primer (first PCR): p1 (SEQ ID NO: 52)
Generated fragment size: 230bp
Primer (second PCR): p2 (SEQ ID NO: 53)
Restriction endonuclease: HinDIII, Tth111I
Cut fragment size: 389bp

[0125]
Mutant: BM11 (N102D)
Mutation-generating oligonucleotide: bm11 (SEQ ID NO: 51)
Primer (first PCR): p3 (SEQ ID NO: 54)
Generated fragment size: 577
Primer (second PCR): p4 (SEQ ID NO: 55)
Restriction endonuclease: HinDIII, PvuI
Cut fragment size: 576
Mutation oligonucleotide
bm4 (SEQ ID NO: 47): F290E
Primer 5 'GTG TTT GAC GTC CCG CTT CAT GAG AAT TTA CAG G
bm5 (SEQ ID NO: 48): F290K
Primer 5 'GTG TTT GAC GTC CCG CTT CAT AAG AAT TTA CAG G
bm6 (SEQ ID NO: 49): F290A
Primer 5 'GTG TTT GAC GTC CCG CTT CAT GCC AAT TTA CAG G
bm8 (SEQ ID NO: 50): Q360E
Primer 5 'AGG GAA TCC GGA TAC CCT GAG GTT TTC TAC GG
bm11 (SEQ ID NO: 51): N102D
Primer 5 'GAT GTG GTT TTG GAT CAT AAG GCC GGC GCT GAT G
Other primers
p1: 5 'CTG TTA TTA ATG CCG CCA AAC C (SEQ ID NO: 52)
p2: 5 'G GAA AAG AAA TGT TTA CGG TTG CG (SEQ ID NO: 53)
p3: 5 'G AAA TGA AGC GGA ACA TCA AAC ACG (SEQ ID NO: 54)
p4: 5 'GTA TGA TTT AGG AGA ATT CC (SEQ ID NO: 55)
Example 11
BAN: 1-300 / Termamyl: 301-483 In a variant derived from a parent hybrid alpha amylase consisting of pH Α-amylase activity
Each enzyme solution was measured by Phadebas test (described above). Activity was measured after 15 minutes incubation at 30 ° C. in 50 mM Britton-Robinson buffer adjusted to the pH indicated by NaOH.
NU / mg enzyme
pH wt Q360E F290A F290K F290E N102D
8.0 5300 7800 8300 4200 6600 6200
9.0 1600 2700 3400 2100 1900 1900
[0126]
Cited references
Klein, C., et al., Biochemistry 1992, 31, 8740-8746,
Mizuno, H., et al., J. Mol. Biol. (1993) 234, 1282-1283,
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Lawson, C.L., J. Mol. Biol. (1994) 236, 590-600,
Qian, M., et al., J. Mol. Biol. (1993) 231, 785-799,
Brady, R.L., et al., Acta Crystallogr. Sect. B, 47, 527-535,
Swift, H.J., et al., Acta Crystallogr. Sect. B, 47, 535-544
A. Kadziola, Ph.D. Thesis: “An alpha-amylase from Barley and its Complex with a Substrate Analogue Inhibitor Studied by X-ray Crystallography”, Department of Chemistry University of Copenhagen 1993
MacGregor, EA, Food Hydrocolloids, 1987, Vol. 1, No. 5-6, p. B. Diderichsen and L. Christiansen, Cloning of a maltogenic α-amylase from Bacillus stearothermophilus, FEMS Microbiol. Letters: 56: pp. 53 -60 (1988)
Hudson et al., Practical Immunology, Third edition (1989), Blackwell Scientific Publications,
Sambrook et al.,Molecular Cloning : A Laboratory Manual, 2nd Ed., Cold Spring Harbor, 1989
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Matthes et al.,The EMBO J. Three, 1984, pp. 801-805.
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Hunkapiller et al., 1984, Nature 310: 105-111
R. Higuchi, B. Krummel, and R.K.Saiki (1988) .A general method of in vitro preparation and specific mutagenesis of DNA fragments: study of protein and DNA interactions.Nucl. Acids Res. 16: 7351-7367.
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Gryczan et al., 1978,J. Bacteriol. 134, pp. 318-329.
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Boel et al., 1990,Biochemistry 29, pp. 6244-6249.

[Brief description of the drawings]
FIG. 1 shows an amino acid sequence alignment of six parent Termamyl-like α-amylases. The numbers at the left end indicate each amino acid sequence as follows:
1: SEQ ID NO: 2
2: Kaoamyl
3: SEQ ID NO: 1
4: SEQ ID NO: 5
5: SEQ ID NO: 4
6: SEQ ID NO: 3
FIG. 2 shows temperature activity curves of SP722 (SEQ ID NO: 2) (at pH 9) and B. licheniformis α-amylase (SEQ ID NO: 4) (at pH 7.3).
FIG. 3 shows temperature activity curves of SP690 (SEQ ID NO: 1), SP722 (SEQ ID NO: 2), and B. licheniformis α-amylase (SEQ ID NO: 4) at pH 10.
FIG. 4 shows an alignment of the amino acid sequences of five α-amylases. The numbers at the left end indicate each amino acid sequence as follows:
1: Mouse α-amylase (amyp mouse)
2: Rat α-amylase (amyp rat)
3: Porcine pancreatic α-amylase (PPA) (amyp pig)
4: Human α-amylase (amyp human)
5: A. haloplanctis α-amylase (AHA) (amy altha)
[Sequence Listing]

Claims (17)

A variant of a parent α-amylase, wherein the parent α-amylase has (i) an amino acid sequence selected from the amino acid sequences shown in SEQ ID NO: 1 or 2, or (ii) one or more of the above amino acids Exhibit at least 90% identity with the sequence, wherein the variant has improved Ca at pH 8-10.5 compared to the parent α-amylase ²⁺ A variant, characterized in that it has stability and the variant has α-amylase activity, and the variant comprises a deletion of amino acid residues at positions 183 and 184 and a mutation N195F. A DNA construct comprising a DNA sequence encoding the α-amylase variant of claim 1 . A recombinant expression vector carrying the DNA construct according to claim 2 . A cell transformed with the DNA construct according to claim 2 or the vector according to claim 3 . The cell according to claim 4 , which is a microorganism. The cell according to claim 5 , which is a bacterium or a fungus. The cell according to claim 6 , which is a gram positive bacterium. For washing and / or dishwashing, the use of α-amylase variant according to claim 1. A detergent additive comprising the α-amylase variant according to claim 1 . Detergent additive according to claim 9 , in the form of non-powdered granules, stabilizing liquid or protected enzyme. The detergent additive according to claim 9 , further comprising another enzyme. A detergent composition comprising the α-amylase variant of claim 1 . The detergent composition according to claim 12 , further comprising another enzyme. A detergent composition for manual or automatic dishwashing comprising the α-amylase variant according to claim 1 . The dishwashing detergent composition according to claim 14 , further comprising another enzyme. A composition for manual or automatic washing and washing comprising the α-amylase variant according to claim 1 . The composition for washing and washing according to claim 16 , further comprising another enzyme.

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